Cell Signalling Through Covalent Modification and Allostery

NASA Astrophysics Data System (ADS)

Johnson, Louise N.

Phosphorylation plays essential roles in nearly every aspect of cell life. Protein kinases catalyze the transfer of the γ-phosphate of ATP to a serine, threonine or tyrosine residue in protein substrates. This covalent modification allows activation or inhibition of enzyme activity, creates recognition sites for other proteins and promotes order/disorder or disorder/order transitions. These properties regulate ­signalling pathways and cellular processes that mediate metabolism, transcription, cell cycle progression, differentiation, cytoskeleton arrangement and cell movement, apoptosis, intercellular communication, and neuronal and immunological functions. In this lecture I shall review the structural consequences of protein phosphorylation using our work on glycogen phosphorylase and the cell cycle cyclin dependent protein kinases as illustrations. Regulation of protein phosphorylation may be disrupted in the diseased state and protein kinases have become high profile targets for drug development. To date there are 11 compounds that have been approved for clinical use in the treatment of cancer.

Cis-existence of H3K27me3 and H3K36me2 in mouse embryonic stem cells revealed by specific ions of isobaric modification chromatogram.

PubMed

Mao, Hailei; Han, Gang; Xu, Longyong; Zhu, Duming; Lin, Hanqing; Cao, Xiongwen; Yu, Yi; Chen, Charlie Degui

2015-07-21

Histone H3 lysine 27 trimethylation (H3K27me3) and H3 lysine 36 trimethylation (H3K36me3) are important epigenetic modifications correlated with transcription repression and activation, respectively. These two opposing modifications rarely co-exist in the same H3 polypeptide. However, a small but significant amount of H3 tails are modified with 5 methyl groups on K27 and K36 in mouse embryonic stem cells (mESCs) and it is unclear how the trimethylation is distributed on K27 or K36. A label-free, bottom-up mass spectrum method, named specific ions of isobaric modification chromatogram (SIMC), was established to quantify the relative abundance of K27me2-K36me3 and K27me3-K36me2 in the same histone H3 tail. By using this method, we demonstrated that the H3K27me3-K36me2 comprises about 85 % of the penta-methylated H3 tails at K27 and K36 in mESCs. Upon mESC differentiation, the abundance of H3K27me3-K36me2 significantly decreased, while the level of H3K27me2-K36me3 remains unchanged. Our study not only revealed the cis-existence of H3K27me3-K36me2 in mESCs, but also suggested that this combinatorial histone modification may assume a specific regulatory function during differentiation.

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

PubMed

Mohr, S; Stamler, J S; Brüne, B

1994-07-18

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.

Efficient Covalent Modification of Multiwalled Carbon Nanotubes with Diazotized Dyes in Water at Room Temperature.

PubMed

Bensghaïer, Asma; Lau Truong, Stéphanie; Seydou, Mahamadou; Lamouri, Aazdine; Leroy, Eric; Mičušik, Matej; Forro, Klaudia; Beji, Mohamed; Pinson, Jean; Omastová, Mária; Chehimi, Mohamed M

2017-07-11

Tetrafluoroborate salts of diazotized Azure A (AA-N 2 + ), Neutral Red (NR-N 2 + ) and Congo Red (CR-N 2 + ) dyes were prepared and reacted with multiwalled carbon nanotubes (MWCNTs) at room temperature, in water without any reducing agent. The as-modified MWCNTs were examined by IRATR, Raman spectroscopy, XPS, TGA, TEM, and cyclic voltammetry. The diazonium band located at ∼2350 cm -1 in the diazotized dye IR spectra vanished after attachment to the nanotubes whereas the Raman D/G peak ratio slightly increased after dye covalent attachment at a high initial diazonium/CNT mass ratio. XPS measurements show the loss of F 1s from the BF 4 - anion together with a clear change in the high-resolution C 1s region from the modified nanotubes. Thermogravimetric analyses proved substantial mass loadings of the organic grafts leveling off at 40.5, 34.3, and 50.7 wt % for AA, NR, and CR, respectively. High-resolution TEM pictures confirmed the presence of 1.5-7-nm-thick continuous amorphous layers on the nanotubes assigned to the aryl layers from the dyes. Cyclic voltammetry studies in acetonitrile (ACN) confirmed the grafting of the dyes; the latter retain their electrochemical behavior in the grafted state. The experimental results correlate remarkably well with quantum chemical calculations that indicate high binding energies between the dyes and the CNTs accounting for true covalent bonding (140-185 kJ/mol with the CNT-aryl distance <1.6 nm), though attachment by π stacking also contributes to obtaining stable hybrids. Finally, the pH-responsive character of the robust hybrids was demonstrated by a higher degree of protonation of Neutral Red-grafted CNTs at pH 2 compared to that of the neutral aqueous medium. This work demonstrates that diazotized dyes can be employed for the surface modification of MWCNTs in a very simple and efficient manner in water and at room temperature. The hybrids could be employed for many purposes such as optically pH-responsive materials

Direct covalent modification as a strategy to inhibit nuclear factor-kappa B.

PubMed

Pande, Vineet; Sousa, Sérgio F; Ramos, Maria João

2009-01-01

Nuclear Factor-KkappaB (NF-kappaB) is a transcription factor whose inappropriate activation may result in the development of a number of diseases including cancer, inflammation, neurodegeneration and AIDS. Recent studies on NF-kappaB mediated pathologies, made therapeutic interventions leading to its inhibition an emerging theme in pharmaceutical research. NF-kappaB resides in the cytoplasm and is activated by several time-dependent factors, leading to proteasome-dependent degradation of its inhibitory protein (IkappaB), resulting in free NF-kappaB (p50 and p65 subunits, involved in disease states), which binds to target DNA sites, further resulting in enhanced transcription of several disease associated proteins. The complex pathway of NF-kappaB, finally leading to its DNA binding, has attracted several approaches interfering with this pathway. One such approach is that of a direct covalent modification of NF-kappaB. In this article, we present a critical review on the pharmacological agents that have been studied as inhibitors of NF-kappaB by covalently modifying redox-regulated cysteine residues in its subunits, ultimately resulting in the inhibition of kappaB DNA recognition and binding. Beginning with a general overview of NF-kappaB pathway and several possibilities of chemical interventions, the significance of redox-regulation in NF-kappaB activation and DNA binding is presented. Further, protein S-thiolation, S-nitrosylation and irreversible covalent modification are described as regular biochemical events in the cell, having provided a guideline for the development of NF-kappaB inhibitors discussed further. Although just a handful of inhibitors, with most of them being alkylating agents have been studied in the present context, this approach presents potential for the development of a new class of NF-kappaB-inhibitors.

Global regulation of post-translational modifications on core histones.

PubMed

Galasinski, Scott C; Louie, Donna F; Gloor, Kristen K; Resing, Katheryn A; Ahn, Natalie G

2002-01-25

Full-length masses of histones were analyzed by mass spectrometry to characterize post-translational modifications of bulk histones and their changes induced by cell stimulation. By matching masses of unique peptides with full-length masses, H4 and the variants H2A.1, H2B.1, and H3.1 were identified as the main histone forms in K562 cells. Mass changes caused by covalent modifications were measured in a dose- and time-dependent manner following inhibition of phosphatases by okadaic acid. Histones H2A, H3, and H4 underwent changes in mass consistent with altered acetylation and phosphorylation, whereas H2B mass was largely unchanged. Unexpectedly, histone H4 became almost completely deacetylated in a dose-dependent manner that occurred independently of phosphorylation. Okadaic acid also partially blocked H4 hyperacetylation induced by trichostatin-A, suggesting that the mechanism of deacetylation involves inhibition of H4 acetyltransferase activity, following perturbation of cellular phosphatases. In addition, mass changes in H3 in response to okadaic acid were consistent with phosphorylation of methylated, acetylated, and phosphorylated forms. Finally, kinetic differences were observed with respect to the rate of phosphorylation of H2A versus H4, suggesting differential regulation of phosphorylation at sites on these proteins, which are highly related by sequence. These results provide novel evidence that global covalent modifications of chromatin-bound histones are regulated through phosphorylation-dependent mechanisms.

Nanoporous Anodic Alumina Surface Modification by Electrostatic, Covalent, and Immune Complexation Binding Investigated by Capillary Filling.

PubMed

Eckstein, Chris; Acosta, Laura K; Pol, Laura; Xifré-Pérez, Elisabet; Pallares, Josep; Ferré-Borrull, Josep; Marsal, Lluis F

2018-03-28

The fluid imbibition-coupled laser interferometry (FICLI) technique has been applied to detect and quantify surface changes and pore dimension variations in nanoporous anodic alumina (NAA) structures. FICLI is a noninvasive optical technique that permits the determination of the NAA average pore radius with high accuracy. In this work, the technique is applied after each step of different surface modification paths of the NAA pores: (i) electrostatic immobilization of bovine serum albumin (BSA), (ii) covalent attachment of streptavidin via (3-aminipropyl)-triethoxysilane and glutaraldehyde grafting, and (iii) immune complexation. Results show that BSA attachment can be detected as a reduction in estimated radius from FICLI with high accuracy and reproducibility. In the case of the covalent attachment of streptavidin, FICLI is able to recognize a multilayer formation of the silane and the protein. For immune complexation, the technique is able to detect different antibody-antigen bindings and distinguish different dynamics among different immune species.

Modeling covalent-modifier drugs.

PubMed

Awoonor-Williams, Ernest; Walsh, Andrew G; Rowley, Christopher N

2017-11-01

In this review, we present a summary of how computer modeling has been used in the development of covalent-modifier drugs. Covalent-modifier drugs bind by forming a chemical bond with their target. This covalent binding can improve the selectivity of the drug for a target with complementary reactivity and result in increased binding affinities due to the strength of the covalent bond formed. In some cases, this results in irreversible inhibition of the target, but some targeted covalent inhibitor (TCI) drugs bind covalently but reversibly. Computer modeling is widely used in drug discovery, but different computational methods must be used to model covalent modifiers because of the chemical bonds formed. Structural and bioinformatic analysis has identified sites of modification that could yield selectivity for a chosen target. Docking methods, which are used to rank binding poses of large sets of inhibitors, have been augmented to support the formation of protein-ligand bonds and are now capable of predicting the binding pose of covalent modifiers accurately. The pK a 's of amino acids can be calculated in order to assess their reactivity towards electrophiles. QM/MM methods have been used to model the reaction mechanisms of covalent modification. The continued development of these tools will allow computation to aid in the development of new covalent-modifier drugs. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of histone modifications in plant leaves.

PubMed

Jaskiewicz, Michal; Peterhansel, Christoph; Conrath, Uwe

2011-09-23

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles(1-2). H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues(1-2). These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)(3-7). Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde(8,9), extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies(9,10), de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C(4;) photosynthesis in maize(5,11) and systemic immunity in Arabidopsis(3).

[Distributions of H3K27me3 and its modification enzymes in different tissues of mice].

PubMed

Wang, Yuying; Wang, Xinli; Zhang, Ran; Zhang, Zhiyan; Wang, Yu; Yang, Bo; Wang, Guanjie; Zhang, Xin; Ma, Fuhao; Xu, Hongye; Wu, Xiaohui; Zhang, Feng; Li, Qing

2017-11-01

Objective To investigate the levels of trimethylated histone 3 at lysine residue 27 (H3K27me3) and its modification enzymes Zeste gene enhancer homolog 2 (EZH2), lysine-specific demethylase 6B (Kdm6B/JMJD3) and lysine-specific demethylase 6A (Kdm6A/UTX) in tissues and organs of 7-day and 2-month postnatal mice. Methods Immunohistochemistry was used to detect the expressions of H3K27me3 and its modification enzymes EZH2, JMJD3 and UTX in the brain, salivary glands, back fat, thymus, lung, heart, stomach, intestines, liver, testes, and skin of 7-day and 2-month mice. Real-time quantitative PCR was used to confirm the results. The relationships between H3K27me3 and its modification enzymes were analyzed statistically. Results Immunohistochemistry showed H3K27me3 persistently present in all examined tissues of 7-day and 2-month mice. EZH2 was persistently expressed in the brain, heart, liver, and skin of 7-day and 2-month mice, but only expressed in the salivary glands, adipose tissues, thymus, lung, intestines, and testes of 2-month mice. JMJD3 was expressed in the brain, salivary glands, adipose tissues, lung, heart, stomach, intestines, testes, skin of 7-day mice, but was not expressed in the lung, adipose tissues and stomach of 2-month mice. UTX was expressed in the brain, salivary glands, adipose tissues, lung, heart, testes, skin of 7-day mice, but only expressed in the testes of 2-month mice. Most mRNA of H3K27 modification enzymes were moderately or highly expressed as their immunohistochemical results were positive. Conclusion There was H3K27me3 persistently present in the all examined tissues at different stages. EZH2 was mostly expressed in the brain, salivary glands, adipose tissues, thymus, lung, heart, intestines, liver, testes and skin of 2-month-old mice. JMJD3 and UTX were mostly expressed in the brain, salivary glands, adipose tissues, lung, heart, skin and testes of 7-day-old mice. No significant association was found between the distribution of H3K

Atom-scale covalent electrochemical modification of single-layer graphene on SiC substrates by diaryliodonium salts

DOE PAGES

Gearba, Raluca I.; Mueller, Kory M.; Veneman, Peter A.; ...

2015-05-09

Owing to its high conductivity, graphene holds promise as an electrode for energy devices such as batteries and photovoltaics. However, to this end, the work function and doping levels in graphene need to be precisely tuned. One promising route for modifying graphene’s electronic properties is via controlled covalent electrochemical grafting of molecules. We show that by employing diaryliodonium salts instead of the commonly used diazonium salts, spontaneous functionalization is avoided. This then allows for precise tuning of the grafting density. Moreover, by employing bis(4-nitrophenyl)iodonium(III) tetrafluoroborate (DNP) salt calibration curves, the surface functionalization density (coverage) of glassy carbon was controlled usingmore » cyclic voltammetry in varying salt concentrations. These electro-grafting conditions and calibration curves translated directly over to modifying single layer epitaxial graphene substrates (grown on insulating 6H-SiC (0 0 0 1)). In addition to quantifying the functionalization densities using electrochemical methods, samples with low grafting densities were characterized by low-temperature scanning tunneling microscopy (LT-STM). We show that the use of buffer-layer free graphene substrates is required for clear observation of the nitrophenyl modifications. Furthermore, atomically-resolved STM images of single site modifications were obtained, showing no preferential grafting at defect sites or SiC step edges as supposed previously in the literature. Most of the grafts exhibit threefold symmetry, but occasional extended modifications (larger than 4 nm) were observed as well.« less

Covalent Surface Modification of Gallium Arsenide Photocathodes for Water Splitting in Highly Acidic Electrolyte.

PubMed

Garner, Logan E; Steirer, K Xerxes; Young, James L; Anderson, Nicholas C; Miller, Elisa M; Tinkham, Jonathan S; Deutsch, Todd G; Sellinger, Alan; Turner, John A; Neale, Nathan R

2017-02-22

Efficient water splitting using light as the only energy input requires stable semiconductor electrodes with favorable energetics for the water-oxidation and proton-reduction reactions. Strategies to tune electrode potentials using molecular dipoles adsorbed to the semiconductor surface have been pursued for decades but are often based on weak interactions and quickly react to desorb the molecule under conditions relevant to sustained photoelectrolysis. Here, we show that covalent attachment of fluorinated, aromatic molecules to p-GaAs(1 0 0) surfaces can be employed to tune the photocurrent onset potentials of p-GaAs(1 0 0) photocathodes and reduce the external energy required for water splitting. Results indicate that initial photocurrent onset potentials can be shifted by nearly 150 mV in pH -0.5 electrolyte under 1 Sun (1000 W m -2 ) illumination resulting from the covalently bound surface dipole. Though X-ray photoelectron spectroscopy analysis reveals that the covalent molecular dipole attachment is not robust under extended 50 h photoelectrolysis, the modified surface delays arsenic oxide formation that results in a p-GaAs(1 0 0) photoelectrode operating at a sustained photocurrent density of -20.5 mA cm -2 within -0.5 V of the reversible hydrogen electrode. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Two supramolecular complexes based on polyoxometalates and Co-EDTA units via covalent connection or non-covalent interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teng, Chunlin; Xiao, Hanxi; Cai, Qing

Two new 3D network organic-inorganic hybrid supramolecular complexes ([Na{sub 6}(CoEDTA){sub 2}(H{sub 2}O){sub 13}]·(H{sub 2}SiW{sub 12}O{sub 40})·xH{sub 2}O)n (1) and [CoH{sub 4}EDTA(H{sub 2}O)]{sub 2}(SiW{sub 12}O{sub 40})·15H{sub 2}O (2) (H{sub 4}EDTA=Ethylenediamine tetraacetic acid) have been successfully synthesized by solution method, and characterized by infrared spectrum (IR), thermogravimetric-differential thermal analysis (TG-DTA), cyclic voltammetry (CV) and single{sup −}crystal X-ray diffraction (XRD). Both of the complexes are the supramolecules, but with different liking mode, they are two representative models of supramolecule. complex (1) is a 3D infinite network supramolecular coordination polymer with a rare multi-metal sturcture of sodium-cobalt-containing, which is mainly linked through coordinate-covalent bonds.more » While complex (2) is normal supramolecule, which linked by non-covalent interactions, such as H-bonding interaction, electrostatic interaction and van der waals force. Both of complex (1) and (2) exhibit good catalytic activities for catalytic oxidation of methanol, when the initial concentration of methanol is 3.0 g m{sup −3}, flow rate is 10 mL min{sup −1}, and the quality of catalyst is 0.2 g, for complex (1) and complex (2) the maximum elimination rates of methanol are 85% (150 °C) and 92% (120 °C), respectively. - Graphical abstract: Two new organic-inorganic hybrid supramolecular complexes based on Co-EDTA, and Keggin polyanions have been successfully synthesized with different pH value by solution method. They are attributed to two representative models of supramolecule. Complex(1) is an infinite coordination polymer with a rare multi-metal sturcture of sodium-cobalt-containing, which is mainly linked through covalent bonds. Complex (2) is a normal supramolecule, which linked by non-covalent interactions of H-bonding interaction, electrostatic interaction and van der waals force. - Highlights: • Two

Covalent modification of a ten-residue cationic antimicrobial peptide with levofloxacin

NASA Astrophysics Data System (ADS)

Rodriguez, Carlos; Papanastasiou, Emilios; Juba, Melanie; Bishop, Barney

2014-09-01

The rampant spread of antibiotic resistant bacteria has spurred interest in alternative strategies for developing next-generation antibacterial therapies. As such, there has been growing interest in cationic antimicrobial peptides (CAMPs) and their therapeutic applications. Modification of CAMPs via conjugation to auxiliary compounds, including small molecule drugs, is a new approach to developing effective, broad-spectrum antibacterial agents with novel physicochemical properties and versatile antibacterial mechanisms. Here, we’ve explored design parameters for engineering CAMPs conjugated to small molecules with favorable physicochemical and antibacterial properties by covalently affixing a fluoroquinolone antibiotic, levofloxacin, to the ten-residue CAMP Pep-4. Relative to the unmodified Pep-4, the conjugate was found to demonstrate substantially increased antibacterial potency under high salt concentrations. Historically, it has been observed that most CAMPs lose antibacterial effectiveness in such high ionic strength environments, a fact that has presented a challenge to their development as therapeutics. Physicochemical studies revealed that P4LC was more hydrophobic than Pep-4, while mechanistic findings indicated that the conjugate was more effective at disrupting bacterial membrane integrity. Although the inherent antibacterial effect of the incorporated levofloxacin molecules did not appear to be substantially realized in this conjugate, these findings nevertheless suggest that covalent attachment of small molecule antibiotics with favorable physicochemical properties to CAMPs could be a promising strategy for enhancing peptide performance and overall therapeutic potential. These results have broader applicability to the development of future CAMP-antibiotic conjugates for potential therapeutic applications.

Covalent surface modification of gallium arsenide photocathodes for water splitting in highly acidic electrolyte

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garner, Logan E.; Steirer, K. Xerxes; Young, James L.

Efficient water splitting using light as the only energy input requires stable semiconductor electrodes with favorable energetics for the water-oxidation and proton-reduction reactions. Strategies to tune electrode potentials using molecular dipoles adsorbed to the semiconductor surface have been pursued for decades but are often based on weak interactions and quickly react to desorb the molecule under conditions relevant to sustained photoelectrolysis. Here, we show that covalent attachment of fluorinated, aromatic molecules to p-GaAs(1 0 0) surfaces can be employed to tune the photocurrent onset potentials of p-GaAs(1 0 0) photocathodes and reduce the external energy required for water splitting. Resultsmore » indicate that initial photocurrent onset potentials can be shifted by nearly 150 mV in pH -0.5 electrolyte under 1 Sun (1000 W m -2) illumination resulting from the covalently bound surface dipole. Furthermore, X-ray photoelectron spectroscopy analysis reveals that the covalent molecular dipole attachment is not robust under extended 50 h photoelectrolysis, the modified surface delays arsenic oxide formation that results in a p-GaAs(1 0 0) photoelectrode operating at a sustained photocurrent density of -20.5 mA cm -2 within -0.5 V of the reversible hydrogen electrode.« less

Covalent surface modification of gallium arsenide photocathodes for water splitting in highly acidic electrolyte

DOE PAGES

Garner, Logan E.; Steirer, K. Xerxes; Young, James L.; ...

2016-12-12

Efficient water splitting using light as the only energy input requires stable semiconductor electrodes with favorable energetics for the water-oxidation and proton-reduction reactions. Strategies to tune electrode potentials using molecular dipoles adsorbed to the semiconductor surface have been pursued for decades but are often based on weak interactions and quickly react to desorb the molecule under conditions relevant to sustained photoelectrolysis. Here, we show that covalent attachment of fluorinated, aromatic molecules to p-GaAs(1 0 0) surfaces can be employed to tune the photocurrent onset potentials of p-GaAs(1 0 0) photocathodes and reduce the external energy required for water splitting. Resultsmore » indicate that initial photocurrent onset potentials can be shifted by nearly 150 mV in pH -0.5 electrolyte under 1 Sun (1000 W m -2) illumination resulting from the covalently bound surface dipole. Furthermore, X-ray photoelectron spectroscopy analysis reveals that the covalent molecular dipole attachment is not robust under extended 50 h photoelectrolysis, the modified surface delays arsenic oxide formation that results in a p-GaAs(1 0 0) photoelectrode operating at a sustained photocurrent density of -20.5 mA cm -2 within -0.5 V of the reversible hydrogen electrode.« less

Real-Space Bonding Indicator Analysis of the Donor-Acceptor Complexes X3BNY3, X3AlNY3, X3BPY3, and X3AlPY3 (X, Y = H, Me, Cl).

PubMed

Mebs, Stefan; Beckmann, Jens

2017-10-12

Calculations of real-space bonding indicators (RSBI) derived from Atoms-In-Molecules (AIM), Electron Localizability Indicator (ELI-D), Non-Covalent Interactions index (NCI), and Density Overlap Regions Indicator (DORI) toolkits for a set of 36 donor-acceptor complexes X 3 BNY 3 (1, 1a-1h), X 3 AlNY 3 (2, 2a-2h), X 3 BPY 3 (3, 3a-3h), and X 3 AlPY 3 (4, 4a-4h) reveal that the donor-acceptor bonds comprise covalent and ionic interactions in varying extents (X = Y = H for 1-4; X = H, Y = Me for 1a-4a; X = H, Y = Cl for 1b-4b; X = Me, Y = H for 1c-4c; X, Y = Me for 1d-4d; X = Me, Y = Cl for 1e-4e; X = Cl, Y = H for 1f-4f; X = Cl, Y = Me for 1g-4g; X, Y = Cl for 1h-4h). The phosphinoboranes X 3 BPY 3 (3, 3a-3h) in general and Cl 3 BPMe 3 (3f) in particular show the largest covalent contributions and the least ionic contributions. The aminoalanes X 3 AlNY 3 (2, 2a-2h) in general and Me 3 AlNCl 3 (2e) in particular show the least covalent contributions and the largest ionic contributions. The aminoboranes X 3 BNY 3 (1, 1a-1h) and the phosphinoalanes X 3 AlPY 3 (4, 4a-4h) are midway between phosphinoboranes and aminoalanes. The degree of covalency and ionicity correlates with the electronegativity difference BP (ΔEN = 0.15) < AlP (ΔEN = 0.58) < BN (ΔEN = 1.00) < AlN (ΔEN = 1.43) and a previously published energy decomposition analysis (EDA). To illustrate the importance of both contributions in Lewis formula representations, two resonance formulas should be given for all compounds, namely, the canonical form with formal charges denoting covalency and the arrow notation pointing from the donor to the acceptor atom to emphasis ionicity. If the Lewis formula mainly serves to show the atomic connectivity, the most significant should be shown. Thus, it is legitimate to present aminoalanes using arrows; however, for phosphinoboranes the canonical form with formal charges is more appropriate.

Dynamic covalent polymers

PubMed Central

García, Fátima

2016-01-01

ABSTRACT This Highlight presents an overview of the rapidly growing field of dynamic covalent polymers. This class of polymers combines intrinsic reversibility with the robustness of covalent bonds, thus enabling formation of mechanically stable, polymer‐based materials that are responsive to external stimuli. It will be discussed how the inherent dynamic nature of the dynamic covalent bonds on the molecular level can be translated to the macroscopic level of the polymer, giving access to a range of applications, such as stimuli‐responsive or self‐healing materials. A primary distinction will be made based on the type of dynamic covalent bond employed, while a secondary distinction will be based on the consideration whether the dynamic covalent bond is used in the main chain of the polymer or whether it is used to allow side chain modification of the polymer. Emphasis will be on the chemistry of the dynamic covalent bonds present in the polymer, in particular in relation to how the specific (dynamic) features of the bond impart functionality to the polymer material, and to the conditions under which this dynamic behavior is manifested. © 2016 The Authors. Journal of Polymer Science Part A: Polymer Chemistry Published by Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016, 54, 3551–3577. PMID:27917019

New fluorescent pH sensors based on covalently linkable PET rhodamines

PubMed Central

Aigner, Daniel; Borisov, Sergey M.; Orriach Fernández, Francisco J.; Fernández Sánchez, Jorge F.; Saf, Robert; Klimant, Ingo

2012-01-01

A new class of rhodamines for the application as indicator dyes in fluorescent pH sensors is presented. Their pH-sensitivity derives from photoinduced electron transfer between non-protonated amino groups and the excited chromophore which results in effective fluorescence quenching at increasing pH. The new indicator class carries a pentafluorophenyl group at the 9-position of the xanthene core where other rhodamines bear 2-carboxyphenyl substituents instead. The pentafluorophenyl group is used for covalent coupling to sensor matrices by “click” reaction with mercapto groups. Photophysical properties are similar to “classical” rhodamines carrying 2′-carboxy groups. pH sensors have been prepared with two different matrix materials, silica gel and poly(2-hydroxyethylmethacrylate). Both sensors show high luminescence brightness (absolute fluorescence quantum yield ΦF≈0.6) and high pH-sensitivity at pH 5–7 which makes them suitable for monitoring biotechnological samples. To underline practical applicability, a dually lifetime referenced sensor containing Cr(III)-doped Al2O3 as reference material is presented. PMID:22967541

Elastomeric nanoparticle composites covalently bound to Al2O3/GaAs surfaces.

PubMed

Song, Hyon Min; Ye, Peide D; Ivanisevic, Albena

2007-08-28

This article reports the modification of Al2O3/GaAs surfaces with multifunctional soft materials. Siloxane elastomers were covalently bound to dopamine-modified Al2O3/GaAs semiconductor surfaces using MPt (M = Fe, Ni) nanoparticles. The sizes of the monodisperse FePt and NiPt nanoparticles were less than 5 nm. The surfaces of the nanoparticles as well as the Al2O3/GaAs substrates were modified with allyl-functionalized dopamine that utilized a dihydroxy group as a strong ligand. The immobilization of the elastomers was performed via a hydrosilation reaction of the allyl-functionalized dopamines with the siloxane backbones. X-ray photoelectron spectroscopy (XPS) experiments confirmed the covalent bonding of the siloxane elastomers to the oxide layer on the semiconductor surface. Fourier transform-infrared reflection absorption spectroscopy (FT-IRRAS) measurements revealed that the allyl functional groups are bonded to the siloxane backbones. The FT-IRRAS data also showed that the density of the allyl groups on the surface was lower than that of the siloxane backbones. The mechanical properties of the surface-bound nanocomposites were tested using nanoindentation experiments. The nanoindentation data showed that the soft matrix composed of the elastomeric coating on the surfaces behaves differently from the inner, hard Al2O3/GaAs substrate.

Sulforaphane Modifies Histone H3, Unpacks Chromatin, and Primes Defense[OPEN

PubMed Central

Jansen, Irina; Baum, Stephani; Beesley, Alexander; Bolm, Carsten

2018-01-01

Modern crop production calls for agrochemicals that prime plants for enhanced defense. Reliable test systems for spotting priming-inducing chemistry, however, are rare. We developed an assay for the high-throughput search for compounds that prime microbial pattern-induced secretion of antimicrobial furanocoumarins (phytoalexins) in cultured parsley cells. The screen produced 1-isothiocyanato-4-methylsulfinylbutane (sulforaphane; SFN), a secondary metabolite in many crucifers, as a novel defense priming compound. While elucidating SFN’s mode of action in defense priming, we found that in Arabidopsis (Arabidopsis thaliana) the isothiocyanate provokes covalent modification (K4me3, K9ac) of histone H3 in the promoter and promoter-proximal region of defense genes WRKY6 and PDF1.2, but not PR1. SFN-triggered H3K4me3 and H3K9ac coincide with chromatin unpacking in the WRKY6 and PDF1.2 regulatory regions, primed WRKY6 expression, unprimed PDF1.2 activation, and reduced susceptibility to downy mildew disease (Hyaloperonospora arabidopsidis). Because SFN also directly inhibits H. arabidopsidis and other plant pathogens, the isothiocyanate is promising for the development of a plant protectant with a dual mode of action. PMID:29288231

Sulforaphane Modifies Histone H3, Unpacks Chromatin, and Primes Defense.

PubMed

Schillheim, Britta; Jansen, Irina; Baum, Stephani; Beesley, Alexander; Bolm, Carsten; Conrath, Uwe

2018-03-01

Modern crop production calls for agrochemicals that prime plants for enhanced defense. Reliable test systems for spotting priming-inducing chemistry, however, are rare. We developed an assay for the high-throughput search for compounds that prime microbial pattern-induced secretion of antimicrobial furanocoumarins (phytoalexins) in cultured parsley cells. The screen produced 1-isothiocyanato-4-methylsulfinylbutane (sulforaphane; SFN), a secondary metabolite in many crucifers, as a novel defense priming compound. While elucidating SFN's mode of action in defense priming, we found that in Arabidopsis ( Arabidopsis thaliana ) the isothiocyanate provokes covalent modification (K4me3, K9ac) of histone H3 in the promoter and promoter-proximal region of defense genes WRKY6 and PDF1 2 , but not PR1 SFN-triggered H3K4me3 and H3K9ac coincide with chromatin unpacking in the WRKY6 and PDF1 2 regulatory regions, primed WRKY6 expression, unprimed PDF1 2 activation, and reduced susceptibility to downy mildew disease ( Hyaloperonospora arabidopsidis ). Because SFN also directly inhibits H arabidopsidis and other plant pathogens, the isothiocyanate is promising for the development of a plant protectant with a dual mode of action. © 2018 American Society of Plant Biologists. All Rights Reserved.

Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor-Electrophile Conjugate.

PubMed

Punthasee, Puminan; Laciak, Adrian R; Cummings, Andrea H; Ruddraraju, Kasi Viswanatharaju; Lewis, Sarah M; Hillebrand, Roman; Singh, Harkewal; Tanner, John J; Gates, Kent S

2017-04-11

Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 10 2 M -1 min -1 . Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) Biochemistry 44, 7704-7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121.

Sulfur mustard-induced epigenetic modifications over time - a pilot study.

PubMed

Simons, Thilo; Steinritz, Dirk; Bölck, Birgit; Schmidt, Annette; Popp, Tanja; Thiermann, Horst; Gudermann, Thomas; Bloch, Wilhelm; Kehe, Kai

2018-09-01

The chemical warfare agent sulfur mustard (SM) can cause long-term health effects that may occur even years after a single exposure. The underlying pathophysiology is unknown, but epigenetic mechanisms are discussed as feasible explanation. "Epigenetics" depicts regulation of gene function without affecting the DNA sequence itself. DNA-methylation and covalent histone modifications (methylation or acetylation) are regarded as important processes. In the present in vitro study using early endothelial cells (EEC), we analyzed SM-induced DNA methylation over time and compared results to an in vivo skin sample that was obtained approx. one year after an accidental SM exposure. EEC were exposed to low SM concentrations (0.5 and 1.0μM). DNA methylation and histone acetylation (H3-K9, H3-K27, H4-K8) or histone di-methylation (H3-K9, H3-K27, H3-K36) were investigated 24h after exposure, and after 2 or 4 additional cell passages. The human skin sample was assessed in parallel. SM had only some minor effects on histone modifications. However, a significant and pronounced increase of DNA methylation was detected in the late cell passages as well as in the skin sample. Our results indicate that SM does indeed cause epigenetic modifications that appear to persist over time. Copyright © 2017 Elsevier B.V. All rights reserved.

Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation

PubMed Central

Grandy, Rodrigo A.; Whitfield, Troy W.; Wu, Hai; Fitzgerald, Mark P.; VanOudenhove, Jennifer J.; Zaidi, Sayyed K.; Montecino, Martin A.; Lian, Jane B.; van Wijnen, André J.; Stein, Janet L.

2015-01-01

Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2, mitotic, and G1 phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotency. PMID:26644406

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells.

PubMed

Malina, Halina Z

2011-01-19

The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells

PubMed Central

2011-01-01

Background The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Results Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. Conclusions The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the

Enhanced H3K4me3 modifications are involved in the transactivation of DNA damage responsive genes in workers exposed to low-level benzene.

PubMed

Li, Jie; Xing, Xiumei; Zhang, Xinjie; Liang, Boxuan; He, Zhini; Gao, Chen; Wang, Shan; Wang, Fangping; Zhang, Haiyan; Zeng, Shan; Fan, Junling; Chen, Liping; Zhang, Zhengbao; Zhang, Bo; Liu, Caixia; Wang, Qing; Lin, Weiwei; Dong, Guanghui; Tang, Huanwen; Chen, Wen; Xiao, Yongmei; Li, Daochuan

2018-03-01

In this study, we explore whether altered global histone modifications respond to low-level benzene exposure as well as their association with the hematotoxicity. We recruited 147 low-level benzene-exposed workers and 122 control workers from a petrochemical factory in Maoming City, Guangdong Province, China. The internal exposure marker level, urinary S-phenylmercapturic acid (SPMA), in benzene-exposed workers was 1.81-fold higher than that of the controls (P < 0.001). ELISA method was established to examine the specific histone modifications in human peripheral blood lymphocytes (PBLCs) of workers. A decrease in the counts of white blood cells (WBC), neutrophils, lymphocytes, and monocytes appeared in the benzene-exposed group (all P < 0.05) compared to the control group. Global trimethylated histone 3 lysine 4 (H3K4me3) modification was enhanced in the benzene-exposed group (P < 0.05) and was positively associated with the concentration of urinary SPMA (β = 0.103, P = 0.045) and the extent of DNA damage (% Tail DNA: β = 0.181, P = 0.022), but was negatively associated with the leukocyte count (WBC: β = -0.038, P = 0.023). The in vitro study revealed that H3K4me3 mark was enriched in the promoters of several DNA damage responsive (DDR) genes including CRY1, ERCC2, and TP53 in primary human lymphocytes treated with hydroquinone. Particularly, H3K4me3 modification was positively correlated with the expression of CRY1 in the PBLCs of benzene-exposed workers. These observations indicate that H3K4me3 modification might mediate the transcriptional regulation of DDR genes in response to low-dose benzene exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hydrides of Alkaline Earth–Tetrel (AeTt) Zintl Phases: Covalent Tt–H Bonds from Silicon to Tin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Auer, Henry; Guehne, Robin; Bertmer, Marko

Zintl phases form hydrides either by incorporating hydride anions (interstitial hydrides) or by covalent bonding of H to the polyanion (polyanionic hydrides), which yields a variety of different compositions and bonding situations. Hydrides (deuterides) of SrGe, BaSi, and BaSn were prepared by hydrogenation (deuteration) of the CrB-type Zintl phases AeTt and characterized by laboratory X-ray, synchrotron, and neutron diffraction, NMR spectroscopy, and quantum-chemical calculations. SrGeD4/3–x and BaSnD4/3–x show condensed boatlike six-membered rings of Tt atoms, formed by joining three of the zigzag chains contained in the Zintl phase. These new polyanionic motifs are terminated by covalently bound H atoms withmore » d(Ge–D) = 1.521(9) Å and d(Sn–D) = 1.858(8) Å. Additional hydride anions are located in Ae4 tetrahedra; thus, the features of both interstitial hydrides and polyanionic hydrides are represented. BaSiD2–x retains the zigzag Si chain as in the parent Zintl phase, but in the hydride (deuteride), it is terminated by H (D) atoms, thus forming a linear (SiD) chain with d(Si–D) = 1.641(5) Å.« less

Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation.

PubMed

Grandy, Rodrigo A; Whitfield, Troy W; Wu, Hai; Fitzgerald, Mark P; VanOudenhove, Jennifer J; Zaidi, Sayyed K; Montecino, Martin A; Lian, Jane B; van Wijnen, André J; Stein, Janet L; Stein, Gary S

2016-02-15

Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2, mitotic, and G1 phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotency. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Synthetic Covalent and Non-Covalent 2D Materials.

PubMed

Boott, Charlotte E; Nazemi, Ali; Manners, Ian

2015-11-16

The creation of synthetic 2D materials represents an attractive challenge that is ultimately driven by their prospective uses in, for example, electronics, biomedicine, catalysis, sensing, and as membranes for separation and filtration. This Review illustrates some recent advances in this diverse field with a focus on covalent and non-covalent 2D polymers and frameworks, and self-assembled 2D materials derived from nanoparticles, homopolymers, and block copolymers. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional crosstalk between histone H2B ubiquitylation and H2A modifications and variants.

PubMed

Wojcik, Felix; Dann, Geoffrey P; Beh, Leslie Y; Debelouchina, Galia T; Hofmann, Raphael; Muir, Tom W

2018-04-11

Ubiquitylation of histone H2B at lysine residue 120 (H2BK120ub) is a prominent histone posttranslational modification (PTM) associated with the actively transcribed genome. Although H2BK120ub triggers several critical downstream histone modification pathways and changes in chromatin structure, less is known about the regulation of the ubiquitylation reaction itself, in particular with respect to the modification status of the chromatin substrate. Here we employ an unbiased library screening approach to profile the impact of pre-existing chromatin modifications on de novo ubiquitylation of H2BK120 by the cognate human E2:E3 ligase pair, UBE2A:RNF20/40. Deposition of H2BK120ub is found to be highly sensitive to PTMs on the N-terminal tail of histone H2A, a crosstalk that extends to the common histone variant H2A.Z. Based on a series of biochemical and cell-based studies, we propose that this crosstalk contributes to the spatial organization of H2BK120ub on gene bodies, and is thus important for transcriptional regulation.

Novel trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-ones act as covalent poisons of human topoisomerase IIα.

PubMed

Infante Lara, Lorena; Sledge, Alexis; Laradji, Amine; Okoro, Cosmas O; Osheroff, Neil

2017-02-01

A number of topoisomerase II-targeted anticancer drugs, including amsacrine, utilize an acridine or related aromatic core as a scaffold. Therefore, to further explore the potential of acridine-related compounds to act as topoisomerase II poisons, we synthesized a series of novel trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-one derivatives and examined their ability to enhance DNA cleavage mediated by human topoisomerase IIα. Derivatives containing a H, Cl, F, and Br at C7 enhanced enzyme-mediated double-stranded DNA cleavage ∼5.5- to 8.5-fold over baseline, but were less potent than amsacrine. The inclusion of an amino group at C9 was critical for activity. The compounds lost their activity against topoisomerase IIα in the presence of a reducing agent, displayed no activity against the catalytic core of topoisomerase IIα, and inhibited DNA cleavage when incubated with the enzyme prior to the addition of DNA. These findings strongly suggest that the compounds act as covalent, rather than interfacial, topoisomerase II poisons. Published by Elsevier Ltd.

Tyrosines of Human and Mouse Transferrin Covalently Labeled by Organophosphorus Agents: A New Motif for Binding to Proteins that Have No Active Site Serine

PubMed Central

Li, Bin; Schopfer, Lawrence M.; Grigoryan, Hasmik; Thompson, Charles M.; Hinrichs, Steven H.; Masson, Patrick; Lockridge, Oksana

2009-01-01

The expectation from the literature is that organophosphorus (OP) agents bind to proteins that have an active site serine. However, transferrin, a protein with no active site serine, was covalently modified in vitro by 0.5mM 10-fluoroethoxyphosphinyl-N-biotinamido pentyldecanamide, chlorpyrifos oxon, diisopropylfluorophosphate, dichlorvos, sarin, and soman. The site of covalent attachment was identified by analyzing tryptic peptides in the mass spectrometer. Tyr 238 and Tyr 574 in human transferrin and Tyr 238, Tyr 319, Tyr 429, Tyr 491, and Tyr 518 in mouse transferrin were labeled by OP. Tyrosine in the small synthetic peptide ArgTyrThrArg made a covalent bond with diisopropylfluorophosphate, chlorpyrifos oxon, and dichlorvos at pH 8.3. These results, together with our previous demonstration that albumin and tubulin bind OP on tyrosine, lead to the conclusion that OP bind covalently to tyrosine, and that OP binding to tyrosine is a new OP-binding residue. The OP-reactive tyrosines are activated by interaction with Arg or Lys. It is suggested that many proteins in addition to those already identified may be modified by OP on tyrosine. The extent to which tyrosine modification by OP can occur in vivo and the toxicological implications of such modifications require further investigation. PMID:18930948

Atomic Covalent Functionalization of Graphene

PubMed Central

Johns, James E.; Hersam, Mark C.

2012-01-01

Conspectus Although graphene’s physical structure is a single atom thick, two-dimensional, hexagonal crystal of sp2 bonded carbon, this simple description belies the myriad interesting and complex physical properties attributed to this fascinating material. Because of its unusual electronic structure and superlative properties, graphene serves as a leading candidate for many next generation technologies including high frequency electronics, broadband photodetectors, biological and gas sensors, and transparent conductive coatings. Despite this promise, researchers could apply graphene more routinely in real-world technologies if they could chemically adjust graphene’s electronic properties. For example, the covalent modification of graphene to create a band gap comparable to silicon (~1 eV) would enable its use in digital electronics, and larger band gaps would provide new opportunities for graphene-based photonics. Towards this end, researchers have focused considerable effort on the chemical functionalization of graphene. Due to its high thermodynamic stability and chemical inertness, new methods and techniques are required to create covalent bonds without promoting undesirable side reactions or irreversible damage to the underlying carbon lattice. In this Account, we review and discuss recent theoretical and experimental work studying covalent modifications to graphene using gas phase atomic radicals. Atomic radicals have sufficient energy to overcome the kinetic and thermodynamic barriers associated with covalent reactions on the basal plane of graphene but lack the energy required to break the C-C sigma bonds that would destroy the carbon lattice. Furthermore, because they are atomic species, radicals substantially reduce the likelihood of unwanted side reactions that confound other covalent chemistries. Overall, these methods based on atomic radicals show promise for the homogeneous functionalization of graphene and the production of new classes of two

A photochemical approach for a fast and self-limited covalent modification of surface supported graphene with photoactive dyes

NASA Astrophysics Data System (ADS)

Sergeeva, Natalia N.; Chaika, Alexander N.; Walls, Brian; Murphy, Barry E.; Walshe, Killian; Martin, David P.; Richards, Billy D. O.; Jose, Gin; Fleischer, Karsten; Aristov, Victor Yu; Molodtsova, Olga V.; Shvets, Igor V.; Krasnikov, Sergey A.

2018-07-01

Herein, we report a simple method for a covalent modification of surface supported graphene with photoactive dyes. Graphene was fabricated on cubic-SiC/Si(001) wafers due to their low cost and suitability for mass-production of continuous graphene fit for electronic applications on millimetre scale. Functionalisation of the graphene surface was carried out in solution via white light induced photochemical generation of phenazine radicals from phenazine diazonium salt. The resulting covalently bonded phenazine-graphene hybrid structure was characterised by scanning tunnelling microscopy (STM) and spectroscopy (STS), Raman spectroscopy and density functional theory (DFT) calculations. It was found that phenazine molecules form an overlayer, which exhibit a short range order with a rectangular unit cell on the graphene surface. DFT calculations based on STM results reveal that molecules are standing up in the overlayer with the maximum coverage of 0.25 molecules per graphene unit cell. Raman spectroscopy and STM results show that the growth is limited to one monolayer of standing molecules. STS reveals that the phenazine-graphene hybrid structure has a band gap of 0.8 eV.

Histone H3 Tails Containing Dimethylated Lysine and Adjacent Phosphorylated Serine Modifications Adopt a Specific Conformation during Mitosis and Meiosis▿ †

PubMed Central

Eberlin, Adrien; Grauffel, Cédric; Oulad-Abdelghani, Mustapha; Robert, Flavie; Torres-Padilla, Maria-Elena; Lambrot, Romain; Spehner, Danièle; Ponce-Perez, Lourdes; Würtz, Jean-Marie; Stote, Roland H.; Kimmins, Sarah; Schultz, Patrick; Dejaegere, Annick; Tora, Laszlo

2008-01-01

Condensation of chromatin, mediated in part by posttranslational modifications of histones, is essential for cell division during mitosis. Histone H3 tails are dimethylated on lysine (Kme2) and become phosphorylated on serine (Sp) residues during mitosis. We have explored the possibility that these double modifications are involved in the establishment of H3 tail conformations during the cell cycle. Here we describe a specific chromatin conformation occurring at Kme2 and adjacently phosphorylated S of H3 tails upon formation of a hydrogen bond. This conformation appears exclusively between early prophase and early anaphase of the mitosis, when chromatin condensation is highest. Moreover, we observed that the conformed H3Kme2Sp tail is present at the diplotene and metaphase stages in spermatocytes and oocytes. Our data together with results obtained by cryoelectron microscopy suggest that the conformation of Kme2Sp-modified H3 tails changes during mitosis and meiosis. This is supported by biostructural modeling of a modified histone H3 tail bound by an antibody, indicating that Kme2Sp-modified H3 tails can adopt at least two different conformations. Thus, the H3K9me2S10p and the H3K27me2S28p sites are involved in the acquisition of specific chromatin conformations during chromatin condensation for cell division. PMID:18180282

Preparation and properties of electro-conductive fabrics based on polypyrrole: covalent vs. non-covalent attachment

NASA Astrophysics Data System (ADS)

David, N. C.; Anavi, D.; Milanovich, M.; Popowski, Y.; Frid, L.; Amir, E.

2017-10-01

Electro-conductive fabrics were prepared via in situ oxidative polymerization of pyrrole (Py) in the presence of unmodified and chemically modified cotton fabrics. Chemical modification of cotton fabric was achieved by covalent attachment of a bifunctional linker molecule to the surface of the fabric, followed by incorporation of a monomer unit onto the linker. The fabrics were characterized using Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron spectroscopy, and thermal analysis. Furthermore, the effect of Py concentration on the degree of polypyrrole (PPy) grafting, surface morphology, electrical resistivity, and laundering durability were studied for both types of cotton fabrics. Reductions of several orders of magnitude in surface and volume electrical resistivities were observed for both non-covalently and covalently linked cotton-PPy systems, whereas the effect of covalent pre-treatment of the fabric was stronger at low Py concentration. On the other hand, at higher monomer concentration, the electrical properties and laundering durability of the fabrics we comparable for both unmodified and chemically pre-treated cotton fabrics, indicating that only a small fraction of PPy chains were chemically grafted onto the fabric surface with the majority of the polymer being connected to the fabric through hydrogen bonds.

Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

PubMed Central

Mendoza, Vanessa Leah; Vachet, Richard W.

2009-01-01

For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

Covalent adduct formation between the plasmalogen-derived modification product 2-chlorohexadecanal and phloretin.

PubMed

Üllen, Andreas; Nusshold, Christoph; Glasnov, Toma; Saf, Robert; Cantillo, David; Eibinger, Gerald; Reicher, Helga; Fauler, Günter; Bernhart, Eva; Hallstrom, Seth; Kogelnik, Nora; Zangger, Klaus; Oliver Kappe, C; Malle, Ernst; Sattler, Wolfgang

2015-02-15

Hypochlorous acid added as reagent or generated by the myeloperoxidase (MPO)-H2O2-Cl(-) system oxidatively modifies brain ether-phospholipids (plasmalogens). This reaction generates a sn2-acyl-lysophospholipid and chlorinated fatty aldehydes. 2-Chlorohexadecanal (2-ClHDA), a prototypic member of chlorinated long-chain fatty aldehydes, has potent neurotoxic potential by inflicting blood-brain barrier (BBB) damage. During earlier studies we could show that the dihydrochalcone-type polyphenol phloretin attenuated 2-ClHDA-induced BBB dysfunction. To clarify the underlying mechanism(s) we now investigated the possibility of covalent adduct formation between 2-ClHDA and phloretin. Coincubation of 2-ClHDA and phloretin in phosphatidylcholine liposomes revealed a half-life of 2-ClHDA of approx. 120min, decaying at a rate of 5.9×10(-3)min(-1). NMR studies and enthalpy calculations suggested that 2-ClHDA-phloretin adduct formation occurs via electrophilic aromatic substitution followed by hemiacetal formation on the A-ring of phloretin. Adduct characterization by high-resolution mass spectroscopy confirmed these results. In contrast to 2-ClHDA, the covalent 2-ClHDA-phloretin adduct was without adverse effects on MTT reduction (an indicator for metabolic activity), cellular adenine nucleotide content, and barrier function of brain microvascular endothelial cells (BMVEC). Of note, 2-ClHDA-phloretin adduct formation was also observed in BMVEC cultures. Intraperitoneal application and subsequent GC-MS analysis of brain lipid extracts revealed that phloretin is able to penetrate the BBB of C57BL/6J mice. Data of the present study indicate that phloretin scavenges 2-ClHDA, thereby attenuating 2-ClHDA-mediated brain endothelial cell dysfunction. We here identify a detoxification pathway for a prototypic chlorinated fatty aldehyde (generated via the MPO axis) that compromises BBB function in vitro and in vivo. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights

Covalent adduct formation between the plasmalogen-derived modification product 2-chlorohexadecanal and phloretin

PubMed Central

Üllen, Andreas; Nusshold, Christoph; Glasnov, Toma; Saf, Robert; Cantillo, David; Eibinger, Gerald; Reicher, Helga; Fauler, Günter; Bernhart, Eva; Hallstrom, Seth; Kogelnik, Nora; Zangger, Klaus; Oliver Kappe, C.; Malle, Ernst; Sattler, Wolfgang

2015-01-01

Hypochlorous acid added as reagent or generated by the myeloperoxidase (MPO)-H2O2-Cl− system oxidatively modifies brain ether-phospholipids (plasmalogens). This reaction generates a sn2-acyl-lysophospholipid and chlorinated fatty aldehydes. 2-Chlorohexadecanal (2-ClHDA), a prototypic member of chlorinated long-chain fatty aldehydes, has potent neurotoxic potential by inflicting blood–brain barrier (BBB) damage. During earlier studies we could show that the dihydrochalcone-type polyphenol phloretin attenuated 2-ClHDA-induced BBB dysfunction. To clarify the underlying mechanism(s) we now investigated the possibility of covalent adduct formation between 2-ClHDA and phloretin. Coincubation of 2-ClHDA and phloretin in phosphatidylcholine liposomes revealed a half-life of 2-ClHDA of approx. 120 min, decaying at a rate of 5.9 × 10−3 min−1. NMR studies and enthalpy calculations suggested that 2-ClHDA-phloretin adduct formation occurs via electrophilic aromatic substitution followed by hemiacetal formation on the A-ring of phloretin. Adduct characterization by high-resolution mass spectroscopy confirmed these results. In contrast to 2-ClHDA, the covalent 2-ClHDA-phloretin adduct was without adverse effects on MTT reduction (an indicator for metabolic activity), cellular adenine nucleotide content, and barrier function of brain microvascular endothelial cells (BMVEC). Of note, 2-ClHDA-phloretin adduct formation was also observed in BMVEC cultures. Intraperitoneal application and subsequent GC–MS analysis of brain lipid extracts revealed that phloretin is able to penetrate the BBB of C57BL/6J mice. Data of the present study indicate that phloretin scavenges 2-ClHDA, thereby attenuating 2-ClHDA-mediated brain endothelial cell dysfunction. We here identify a detoxification pathway for a prototypic chlorinated fatty aldehyde (generated via the MPO axis) that compromises BBB function in vitro and in vivo. PMID:25576489

Site-specific covalent modifications of human insulin by catechol estrogens: Reactivity and induced structural and functional changes

NASA Astrophysics Data System (ADS)

Ku, Ming-Chun; Fang, Chieh-Ming; Cheng, Juei-Tang; Liang, Huei-Chen; Wang, Tzu-Fan; Wu, Chih-Hsing; Chen, Chiao-Chen; Tai, Jung-Hsiang; Chen, Shu-Hui

2016-06-01

Proteins, covalently modified by catechol estrogens (CEs), were identified recently from the blood serum of diabetic patients and referred to as estrogenized proteins. Estrogenization of circulating insulin may occur and affect its molecular functioning. Here, the chemical reactivity of CEs towards specific amino acid residues of proteins and the structural and functional changes induced by the estrogenization of insulin were studied using cyclic voltammetry, liquid chromatography-mass spectrometry, circular dichroism spectroscopy, molecular modeling, and bioassays. Our results indicate that CEs, namely, 2- and 4-hydroxyl estrogens, were thermodynamically and kinetically more reactive than the catechol moiety. Upon co-incubation, intact insulin formed a substantial number of adducts with one or multiple CEs via covalent conjugation at its Cys 7 in the A or B chain, as well as at His10 or Lys29 in the B chain. Such conjugation was coupled with the cleavage of inter-chain disulfide linkages. Estrogenization on these sites may block the receptor-binding pockets of insulin. Insulin signaling and glucose uptake levels were lower in MCF-7 cells treated with modified insulin than in cells treated with native insulin. Taken together, our findings demonstrate that insulin molecules are susceptible to active estrogenization, and that such modification may alter the action of insulin.

Design and synthesis of (aza)indolyl maleimide-based covalent inhibitors of glycogen synthase kinase 3β.

PubMed

Yang, Zhimin; Liu, Hui; Pan, Botao; He, Fengli; Pan, Zhengying

2018-05-21

As an important kinase in multiple signal transduction pathways, GSK-3β has been an attractive target for chemical probe discovery and drug development. Compared to numerous reversible inhibitors that have been developed, covalent inhibitors of GSK-3β are noticeably lacking. Here, we report the discovery of a series of covalent GSK-3β inhibitors by optimizing both non-covalent interactions and reactive groups. Among these covalent inhibitors, compound 38b with a mild α-fluoromethyl amide reactive group emerges as a selective and covalent inhibitor against GSK-3β, effectively inhibits the phosphorylation of glycogen synthase and tau protein, and increases β-catenin's levels in living cells. In addition, compound 38b is highly permeable and not a substrate of P-glycoprotein.

Covalent Modification of Highly Ordered Pyrolytic Graphite with a Stable Organic Free Radical by Using Diazonium Chemistry.

PubMed

Seber, Gonca; Rudnev, Alexander V; Droghetti, Andrea; Rungger, Ivan; Veciana, Jaume; Mas-Torrent, Marta; Rovira, Concepció; Crivillers, Núria

2017-01-26

A novel, persistent, electrochemically active perchlorinated triphenylmethyl (PTM) radical with a diazonium functionality has been covalently attached to highly ordered pyrolytic graphite (HOPG) by electrografting in a single-step process. Electrochemical scanning tunneling microscopy (EC-STM) and Raman spectroscopy measurements revealed that PTM molecules had a higher tendency to covalently react at the HOPG step edges. The cross-section profiles from EC-STM images showed that there was current enhancement at the functionalized areas, which could be explained by redox-mediated electron tunneling through surface-confined redox-active molecules. Cyclic voltammetry clearly demonstrated that the intrinsic properties of the organic radical were preserved upon grafting and DFT calculations also revealed that the magnetic character of the PTM radical was preserved. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reversible and formaldehyde-mediated covalent binding of a bis-amino mitoxantrone analogue to DNA.

PubMed

Konda, Shyam K; Kelso, Celine; Pumuye, Paul P; Medan, Jelena; Sleebs, Brad E; Cutts, Suzanne M; Phillips, Don R; Collins, J Grant

2016-05-18

The ability of a bis-amino mitoxantrone anticancer drug (named WEHI-150) to form covalent adducts with DNA, after activation by formaldehyde, has been studied by electrospray ionisation mass spectrometry and HPLC. Mass spectrometry results showed that WEHI-150 could form covalent adducts with d(ACGCGCGT)2 that contained one, two or three covalent links to the octanucleotide, whereas the control drugs (daunorubicin and the anthracenediones mitoxantrone and pixantrone) only formed adducts with one covalent link to the octanucleotide. HPLC was used to examine the extent of covalent bond formation of WEHI-150 with d(CGCGCG)2 and d(CG(5Me)CGCG)2. Incubation of WEHI-150 with d(CG(5Me)CGCG)2 in the presence of formaldehyde resulted in the formation of significantly greater amounts of covalent adducts than was observed with d(CGCGCG)2. In order to understand the observed increase of covalent adducts with d(CG(5Me)CGCG)2, an NMR study of the reversible interaction of WEHI-150 at both CpG and (5Me)CpG sites was undertaken. Intermolecular NOEs were observed in the NOESY spectra of d(ACGGCCGT)2 with added WEHI-150 that indicated that the drug selectively intercalated at the CpG sites and from the major groove. In particular, NOEs were observed from the WEHI-150 H2,3 protons to the H1' protons of G3 and G7 and from the H6,7 protons to the H5 protons of C2 and C6. By contrast, intermolecular NOEs were observed between the WEHI-150 H2,3 protons to the H2'' proton of the (5Me)C3 in d(CG(5Me)CGCG)2, and between the drug aliphatic protons and the H1' proton of G4. This demonstrated that WEHI-150 preferentially intercalates at (5Me)CpG sites, compared to CpG sequences, and predominantly via the minor groove at the (5Me)CpG site. The results of this study demonstrate that WEHI-150 is likely to form interstrand DNA cross-links, upon activation by formaldehyde, and consequently exhibit greater cytotoxicity than other current anthracenedione drugs.

Variation of the chemical reactivity of Thermus thermophilus HB8 ribosomal proteins as a function of pH.

PubMed

Running, William E; Reilly, James P

2010-10-01

Ribosomes occupy a central position in cellular metabolism, converting stored genetic information into active cellular machinery. Ribosomal proteins modulate both the intrinsic function of the ribosome and its interaction with other cellular complexes, such as chaperonins or the signal recognition particle. Chemical modification of proteins combined with mass spectrometric detection of the extent and position of covalent modifications is a rapid, sensitive method for the study of protein structure and flexibility. By altering the pH of the solution, we have induced non-denaturing changes in the structure of bacterial ribosomal proteins and detected these conformational changes by covalent labeling. Changes in ribosomal protein modification across a pH range from 6.6 to 8.3 are unique to each protein, and correlate with their structural environment in the ribosome. Lysine residues whose extent of modification increases as a function of increasing pH are on the surface of proteins, but in close proximity either to glutamate and aspartate residues, or to rRNA backbone phosphates. Increasing pH disrupts tertiary and quaternary interactions mediated by hydrogen bonding or ionic interactions, and regions of protein structure whose conformations are sensitive to these changes are of potential importance in modulating the flexibility of the ribosome or its interaction with other cellular complexes.

Generation of novel covalent RNA-protein complexes in cells by ultraviolet B irradiation: implications for autoimmunity.

PubMed

Andrade, Felipe; Casciola-Rosen, Livia A; Rosen, Antony

2005-04-01

To determine whether ultraviolet B (UVB) irradiation induces novel modifications in autoantigens targeted during experimental photoinduced epidermal damage. To search for novel UVB-induced autoantigen modifications, lysates made from UVB-irradiated human keratinocytes or HeLa cells were immunoblotted using human autoantibodies that recognize ribonucleoprotein autoantigens. Novel autoantigen structures identified were further characterized using nucleases and RNA hybridization. Human sera that recognize U1-70 kd (U1-70K) and La by immunoblotting also recognized multiple novel species when they were used to immunoblot lysates of UVB-irradiated keratinocytes or HeLa cells. These species were not present in control cells and were not observed when apoptosis was induced by Fas ligation or cytotoxic lymphocyte granule contents. Biochemical analysis using multiple assays revealed that these novel UVB-induced molecular species result from the covalent crosslinking between the U1 RNA and the hYRNA molecules with their associated proteins, including U1-70K, La, and likely components of the Sm particle. These data demonstrate that UVB irradiation of live cells can directly induce covalent RNA-protein complexes, which are recognized by human autoantibodies. As previously described for other autoantigens, these covalent complexes of RNA and proteins may have important consequences in terms of antigen capture and processing.

Fast and accurate predictions of covalent bonds in chemical space.

PubMed

Chang, K Y Samuel; Fias, Stijn; Ramakrishnan, Raghunathan; von Lilienfeld, O Anatole

2016-05-07

We assess the predictive accuracy of perturbation theory based estimates of changes in covalent bonding due to linear alchemical interpolations among molecules. We have investigated σ bonding to hydrogen, as well as σ and π bonding between main-group elements, occurring in small sets of iso-valence-electronic molecules with elements drawn from second to fourth rows in the p-block of the periodic table. Numerical evidence suggests that first order Taylor expansions of covalent bonding potentials can achieve high accuracy if (i) the alchemical interpolation is vertical (fixed geometry), (ii) it involves elements from the third and fourth rows of the periodic table, and (iii) an optimal reference geometry is used. This leads to near linear changes in the bonding potential, resulting in analytical predictions with chemical accuracy (∼1 kcal/mol). Second order estimates deteriorate the prediction. If initial and final molecules differ not only in composition but also in geometry, all estimates become substantially worse, with second order being slightly more accurate than first order. The independent particle approximation based second order perturbation theory performs poorly when compared to the coupled perturbed or finite difference approach. Taylor series expansions up to fourth order of the potential energy curve of highly symmetric systems indicate a finite radius of convergence, as illustrated for the alchemical stretching of H2 (+). Results are presented for (i) covalent bonds to hydrogen in 12 molecules with 8 valence electrons (CH4, NH3, H2O, HF, SiH4, PH3, H2S, HCl, GeH4, AsH3, H2Se, HBr); (ii) main-group single bonds in 9 molecules with 14 valence electrons (CH3F, CH3Cl, CH3Br, SiH3F, SiH3Cl, SiH3Br, GeH3F, GeH3Cl, GeH3Br); (iii) main-group double bonds in 9 molecules with 12 valence electrons (CH2O, CH2S, CH2Se, SiH2O, SiH2S, SiH2Se, GeH2O, GeH2S, GeH2Se); (iv) main-group triple bonds in 9 molecules with 10 valence electrons (HCN, HCP, HCAs, HSiN, HSi

Covalent modifications of the amyloid beta peptide by hydroxynonenal: Effects on metal ion binding by monomers and insights into the fibril topology.

PubMed

Grasso, G; Komatsu, H; Axelsen, P H

2017-09-01

Amyloid β peptides (Aβ) and metal ions are associated with oxidative stress in Alzheimer's disease (AD). Oxidative stress, acting on ω-6 polyunsaturated fatty acyl chains, produces diverse products, including 4-hydroxy-2-nonenal (HNE), which can covalently modify the Aβ that helped to produce it. To examine possible feedback mechanisms involving Aβ, metal ions and HNE production, the effects of HNE modification and fibril formation on metal ion binding was investigated. Results indicate that copper(II) generally inhibits the modification of His side chains in Aβ by HNE, but that once modified, copper(II) still binds to Aβ with high affinity. Fibril formation protects only one of the three His residues in Aβ from HNE modification, and this protection is consistent with proposed models of fibril structure. These results provide insight into a network of biochemical reactions that may be operating as a consequence of oxidative stress in AD, or as part of the pathogenic process. Copyright © 2016. Published by Elsevier Inc.

Covalency in Americium(III) Hexachloride

DOE PAGES

Cross, Justin Neil; Su, Jing; Batista, Enrigue R.; ...

2017-06-14

Developing a better understanding of covalency (or orbital mixing) is of fundamental importance. Covalency occupies a central role in directing chemical and physical properties for almost any given compound or material. Hence, the concept of covalency has potential to generate broad and substantial scientific advances, ranging from biological applications to condensed matter physics. Given the importance orbital mixing combined with the difficultly in measuring covalency, estimating or inferring covalency often leads to fiery debate. Consider the 60-year controversy sparked by SEABORG and COWORKERS (1954) when it was proposed that covalency from 5f-orbitals contributed to the unique behavior of americium inmore » chloride matrixes. Herein, we describe the use of ligand K-edge X-ray absorption spectroscopy (XAS) and electronic structure calculations to quantify the extent of covalent bonding in – arguably – one of the most difficult systems to study, the Am–Cl interaction within AmCl 6 3-. We observed both 5fand 6d-orbital mixing with the Cl-3p orbitals; however, contributions from the 6d-orbitals were more substantial. Comparisons with the isoelectronic EuCl 6 3- indicated similar bonding for the Am III 6d- and Eu III 5d-orbitals. Meanwhile, the results confirmed SEABORG’S 1954 hypothesis that Am III 5f-orbital covalency was more substantial than 4forbital mixing for Eu III.« less

Covalent character and electric field dependence of H2-AgX (X = F - I).

PubMed

Li, Xinying

2018-06-16

Mechanisms of Ag-X and Ag…H 2 interactions and stabilities of T-shaped H 2 -AgX (X = F - I) series were investigated at the CCSD(T) level. The "no-density" bond with smaller positive Laplacian and ELF values in interaction regions, as well as considerable delocalization index values, suggest weak covalent "charge-shift" character for the Ag…H 2 interaction. Structure and stability dependence on the electric field were investigated at the MP2 level. Relative total energy curves show obvious parabolic character, and the plots can be fitted by quadratic polynomials as functions of electric field strength.

Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Dan; Hu, Qi; Li, Qing

2013-04-08

Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription. In Saccharomyces cerevisiae, the histone chaperone Rtt106 contributes to the deposition of newly synthesized H3K56ac-carrying H3-H4 complex on replicating DNA, but it is unclear how Rtt106 binds H3-H4 and specifically recognizes H3K56ac as there is no apparent acetylated lysinemore » reader domain in Rtt106. Here, we show that two domains of Rtt106 are involved in a combinatorial recognition of H3-H4. An N-terminal domain homodimerizes and interacts with H3-H4 independently of acetylation while a double pleckstrin-homology (PH) domain binds the K56-containing region of H3. Affinity is markedly enhanced upon acetylation of K56, an effect that is probably due to increased conformational entropy of the αN helix of H3. Our data support a mode of interaction where the N-terminal homodimeric domain of Rtt106 intercalates between the two H3-H4 components of the (H3-H4) 2 tetramer while two double PH domains in the Rtt106 dimer interact with each of the two H3K56ac sites in (H3-H4) 2. We show that the Rtt106-(H3-H4) 2 interaction is important for gene silencing and the DNA damage response.« less

Modification of smoothing in 4253H[T

NASA Astrophysics Data System (ADS)

Azmi, Nurul Nisa'Khairol; Adam, Mohd Bakri; Shitan, Mahendran; Ali, Norhaslinda Mohd

2017-05-01

Some modified non-linear smoothers particularly 4253H[T] are explained in this paper. The modifications are focused on estimating the middle point of running median for even span by applying the following types of means; geometric, harmonic, quadratic and contraharmonic. The performance of the techniques is assessed by applying it to daily price index of a bank in Malaysia that issues sukuk for funding in Islamic banking and financial business. The results show that 4253H[T] with geometric mean modification is better than others in preserving variation and curve fitting.

The Histone Modification H3K27me3 Is Retained after Gene Duplication and Correlates with Conserved Noncoding Sequences in Arabidopsis

PubMed Central

Berke, Lidija; Snel, Berend

2014-01-01

The histone modification H3K27me3 is involved in repression of transcription and plays a crucial role in developmental transitions in both animals and plants. It is deposited by PRC2 (Polycomb repressive complex 2), a conserved protein complex. In Arabidopsis thaliana, H3K27me3 is found at 15% of all genes. These tend to encode transcription factors and other regulators important for development. However, it is not known how PRC2 is recruited to target loci nor how this set of target genes arose during Arabidopsis evolution. To resolve the latter, we integrated A. thaliana gene families with five independent genome-wide H3K27me3 data sets. Gene families were either significantly enriched or depleted of H3K27me3, showing a strong impact of shared ancestry to H3K27me3 distribution. To quantify this, we performed ancestral state reconstruction of H3K27me3 on phylogenetic trees of gene families. The set of H3K27me3-marked genes changed less than expected by chance, suggesting that H3K27me3 was retained after gene duplication. This retention suggests that the PRC2-recruiting signal could be encoded in the DNA and also conserved among certain duplicated genes. Indeed, H3K27me3-marked genes were overrepresented among paralogs sharing conserved noncoding sequences (CNSs) that are enriched with transcription factor binding sites. The association of upstream CNSs with H3K27me3-marked genes represents the first genome-wide connection between H3K27me3 and potential regulatory elements in plants. Thus, we propose that CNSs likely function as part of the PRC2 recruitment in plants. PMID:24567304

Biofunctional Paper via Covalent Modification of Cellulose

PubMed Central

Yu, Arthur; Shang, Jing; Cheng, Fang; Paik, Bradford A.; Kaplan, Justin M.; Andrade, Rodrigo B.; Ratner, Daniel M.

2012-01-01

Paper-based analytical devices are the subject of growing interest for the development of low-cost point-of-care diagnostics, environmental monitoring technologies and research tools for limited-resource settings. However, there are limited chemistries available for the conjugation of biomolecules to cellulose for use in biomedical applications. Herein, divinyl sulfone (DVS) chemistry was demonstrated to covalently immobilize small molecules, proteins and DNA onto the hydroxyl groups of cellulose membranes through nucleophilic addition. Assays on modified cellulose using protein-carbohydrate and protein-glycoprotein interactions as well as oligonucleotide hybridization showed that the membrane’s bioactivity was specific, dose-dependent, and stable over a long period of time. Use of an inkjet printer to form patterns of biomolecules on DVS-activated cellulose illustrates the adaptability of the DVS functionalization technique to pattern sophisticated designs, with potential applications in cellulose-based lateral flow devices. PMID:22708701

Covalent Immobilization of (-)-Riboflavin on Polymer Functionalized Silica Particles: Application in the Photocatalytic E→Z Isomerization of Polarized Alkenes.

PubMed

Metternich, Jan B; Sagebiel, Sven; Lückener, Anne; Lamping, Sebastian; Ravoo, Bart Jan; Gilmour, Ryan

2018-03-20

The covalent immobilization of the biomimetic, photo-organocatalyst (-)-riboflavin on silica micro- and nanoparticles via atom transfer radical polymerization (ATRP) is disclosed. Given the effectiveness of (-)-riboflavin as a versatile, environmentally benign photocatalyst, an immobilization strategy based on acrylate-linker modification of the catalyst core and controlled polymerization on initiator pre-functionalized silica particles has been developed. Validation of this approach is demonstrated in the E→Z isomerization of a benchmark cinnamonitrile (Z/E up to 88:12) with 0.97 mol % catalyst loading. Characterization of the immobilized photocatalyst supports covalent embedding of the catalyst in the polymeric brushes on the silica particle surface. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterizing Covalently Sidewall-Functionalized SWCNTs by using 1H NMR Spectroscopy

PubMed Central

Nelson, Donna J.; Kumar, Ravi

2013-01-01

Unambiguous evidence for covalent sidewall functionalization of single-walled carbon nanotubes (SWCNTs) has been a difficult task, especially for nanomaterials in which slight differences in functionality structure produce significant changes in molecular characteristics. Nuclear magnetic resonance (NMR) spectroscopy provides clear information about the structural skeleton of molecules attached to SWCNTs. In order to establish the generality of proton NMR as an analytical technique for characterizing covalently functionalized SWCNTs, we have obtained and analyzed proton NMR data of SWCNT-substituted benzenes across a variety of para substituents. Trends obtained for differences in proton NMR chemical shifts and the impact of o-, p-, and m-directing effects of electrophilic aromatic substituents on phenyl groups covalently bonded to SWCNTs are discussed. PMID:24009779

Al6H18: A baby crystal of γ-AlH3

NASA Astrophysics Data System (ADS)

Kiran, B.; Kandalam, Anil K.; Xu, Jing; Ding, Y. H.; Sierka, M.; Bowen, K. H.; Schnöckel, H.

2012-10-01

Using global-minima search methods based on the density functional theory calculations of (AlH3)n (n = 1-8) clusters, we show that the growth pattern of alanes for n ≥ 4 is dominated by structures containing hexa-coordinated Al atoms. This is in contrast to the earlier studies where either linear or ring structures of AlH3 were predicted to be the preferred structures in which the Al atoms can have a maximum of five-fold coordination. Our calculations also reveal that the Al6H18 cluster, with its hexa-coordination of the Al atoms, resembles the unit-cell of γ-AlH3, thus Al6H18 is designated as the "baby crystal." The fragmentation energies of the (AlH3)n (n = 2-8) along with the dimerization energies for even n clusters indicate an enhanced stability of the Al6H18 cluster. Both covalent (hybridization) and ionic (charge) contribution to the bonding are the driving factors in stabilizing the isomers containing hexa-coordinated Al atoms.

Non-covalent binding of nucleic acids with gold nanoparticles provides their stability and effective desorption in environment mimicking biological media.

PubMed

Epanchintseva, Anna; Dolodoev, Anton; Grigor'eva, Alina; Chelobanov, Boris; Pyshnyi, Dmitrii; Ryabchikova, Elena; Pyshnaya, Inna

2018-08-31

The ability of gold nanoparticles to bind different substances has resulted in the high interest of researchers determining their usage as a promising carrier of various biological substances including nucleic acids (NAs) for therapeutic applications. Most publications report covalent binding (conjugation) of an NA to spherical AuNPs via the Au-S bond. In this work, we obtained non-covalent associates of different ssDNA, ssRNA and siRNAs with spherical gold nanoparticles (AuNPs) and examined their physico-chemical properties and stability in media mimicking intracellular space (bacterial 'cytosol') and cell culture media (10% FBS in DMEM). The 'cytosol' was obtained from E. coli and possessed nuclease activity. For the first time, we used the phosphoryl guanidine (dimethylimidazolidin-2-imine, Dmi) group for modification of 3'-ends to enhance the stability of ssRNAs and siRNAs against nuclease destruction. Trying to evaluate the material balance, we analyzed the whole nucleotide species obtained after incubation of NA-AuNPs associates in 'cytosol' and FBS and evaluated the degree of NAs destruction, a share of full-size NAs remained on the surface of the AuNPs and in the solution. Native ss- and siRNAs, both free and in composition of non-covalent associates with AuNPs, were less resistant to degrading factors than ssDNA. The introduction of two Dmi-groups into the ssDNA increased its stability in 'cytosol' three times within 2.5 h. Dmi-modified siRNAs in non-covalent associates with AuNPs were two times more stable than unmodified siRNA within 4 h. We showed that non-covalent siRNA-AuNPs associates serve as a kind of storage for full-size NAs and thereby prolong their presence in nuclease-active media. Our study showed that non-covalent binding of siRNAs with a surface of AuNPs provides desorption of both strands, which is necessary for siRNA functioning in living cells, and could be considered as an important way to construct siRNA and ssDNA delivery systems

Discovery of a Chemical Modification by Citric Acid in a Recombinant Monoclonal Antibody

PubMed Central

2015-01-01

Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2 and led to the formation of so-called “acidic species” that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain, or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again reminded us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity. PMID:25136741

Communication: Low-energy free-electron driven molecular engineering: In situ preparation of intrinsically short-lived carbon-carbon covalent dimer of CO

NASA Astrophysics Data System (ADS)

Davis, Daly; Sajeev, Y.

2017-02-01

Molecular modification induced through the resonant attachment of a low energy electron (LEE) is a novel approach for molecular engineering. In this communication, we explore the possibility to use the LEE as a quantum tool for the in situ preparation of short lived molecules. Using ab initio quantum chemical methods, this possibility is best illustrated for the in situ preparation of the intrinsically short-lived carbon-carbon covalent dimer of CO from a glyoxal molecule. The chemical conversion of glyoxal to the covalent dimer of CO is initiated and driven by the resonant capture of a near 11 eV electron by the glyoxal molecule. The resulting two-particle one-hole (2p-1h) negative ion resonant state (NIRS) of the glyoxal molecule undergoes a barrierless radical dehydrogenation reaction and produces the covalent dimer of CO. The autoionization electron spectra from the 2p-1h NIRS at the dissociation limit of the dehydrogenation reaction provides access to the electronic states of the CO dimer. The overall process is an example of a catalytic electron reaction channel.

G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis

PubMed Central

Tachibana, Makoto; Sugimoto, Kenji; Nozaki, Masami; Ueda, Jun; Ohta, Tsutomu; Ohki, Misao; Fukuda, Mikiko; Takeda, Naoki; Niida, Hiroyuki; Kato, Hiroyuki; Shinkai, Yoichi

2002-01-01

Covalent modification of histone tails is crucial for transcriptional regulation, mitotic chromosomal condensation, and heterochromatin formation. Histone H3 lysine 9 (H3-K9) methylation catalyzed by the Suv39h family proteins is essential for establishing the architecture of pericentric heterochromatin. We recently identified a mammalian histone methyltransferase (HMTase), G9a, which has strong HMTase activity towards H3-K9 in vitro. To investigate the in vivo functions of G9a, we generated G9a-deficient mice and embryonic stem (ES) cells. We found that H3-K9 methylation was drastically decreased in G9a-deficient embryos, which displayed severe growth retardation and early lethality. G9a-deficient ES cells also exhibited reduced H3-K9 methylation compared to wild-type cells, indicating that G9a is a dominant H3-K9 HMTase in vivo. Importantly, the loss of G9a abolished methylated H3-K9 mostly in euchromatic regions. Finally, G9a exerted a transcriptionally suppressive function that depended on its HMTase activity. Our results indicate that euchromatic H3-K9 methylation regulated by G9a is essential for early embryogenesis and is involved in the transcriptional repression of developmental genes. PMID:12130538

Beyond cysteine: recent developments in the area of targeted covalent inhibition.

PubMed

Mukherjee, Herschel; Grimster, Neil P

2018-05-29

Over the past decade targeted covalent inhibitors have undergone a renaissance due to the clinical validation and regulatory approval of several small molecule therapeutics that are designed to irreversibly modify their target protein. Invariably, these compounds rely on the serendipitous placement of a cysteine residue proximal to the small molecule binding site; while this strategy has afforded numerous successes, it necessarily limits the number of proteins that can be targeted by this approach. This drawback has led several research groups to develop novel methodologies that target non-cysteine residues for covalent modification. Herein, we survey the current literature of warheads that covalently modify non-cysteine amino acids in proteins. Copyright © 2018 Elsevier Ltd. All rights reserved.

Covalent modification of pericardial patches for sustained rapamycin delivery inhibits venous neointimal hyperplasia

PubMed Central

Bai, Hualong; Lee, Jung Seok; Chen, Elizabeth; Wang, Mo; Xing, Ying; Fahmy, Tarek M.; Dardik, Alan

2017-01-01

Prosthetic grafts and patches are commonly used in cardiovascular surgery, however neointimal hyperplasia remains a significant concern, especially under low flow conditions. We hypothesized that delivery of rapamycin from nanoparticles (NP) covalently attached to patches allows sustained site-specific delivery of therapeutic agents targeted to inhibit localized neointimal hyperplasia. NP were covalently linked to pericardial patches using EDC/NHS chemistry and could deliver at least 360 ng rapamycin per patch without detectable rapamycin in serum; nanoparticles were detectable in the liver, kidney and spleen but no other sites within 24 hours. In a rat venous patch angioplasty model, control patches developed robust neointimal hyperplasia on the patch luminal surface characterized by Eph-B4-positive endothelium and underlying SMC and infiltrating cells such as macrophages and leukocytes. Patches delivering rapamycin developed less neointimal hyperplasia, less smooth muscle cell proliferation, and had fewer infiltrating cells but retained endothelialization. NP covalently linked to pericardial patches are a novel composite delivery system that allows sustained site-specific delivery of therapeutics; NP delivering rapamycin inhibit patch neointimal hyperplasia. NP linked to patches may represent a next generation of tissue engineered cardiovascular implants. PMID:28071663

Covalent modification of pericardial patches for sustained rapamycin delivery inhibits venous neointimal hyperplasia

NASA Astrophysics Data System (ADS)

Bai, Hualong; Lee, Jung Seok; Chen, Elizabeth; Wang, Mo; Xing, Ying; Fahmy, Tarek M.; Dardik, Alan

2017-01-01

Prosthetic grafts and patches are commonly used in cardiovascular surgery, however neointimal hyperplasia remains a significant concern, especially under low flow conditions. We hypothesized that delivery of rapamycin from nanoparticles (NP) covalently attached to patches allows sustained site-specific delivery of therapeutic agents targeted to inhibit localized neointimal hyperplasia. NP were covalently linked to pericardial patches using EDC/NHS chemistry and could deliver at least 360 ng rapamycin per patch without detectable rapamycin in serum; nanoparticles were detectable in the liver, kidney and spleen but no other sites within 24 hours. In a rat venous patch angioplasty model, control patches developed robust neointimal hyperplasia on the patch luminal surface characterized by Eph-B4-positive endothelium and underlying SMC and infiltrating cells such as macrophages and leukocytes. Patches delivering rapamycin developed less neointimal hyperplasia, less smooth muscle cell proliferation, and had fewer infiltrating cells but retained endothelialization. NP covalently linked to pericardial patches are a novel composite delivery system that allows sustained site-specific delivery of therapeutics; NP delivering rapamycin inhibit patch neointimal hyperplasia. NP linked to patches may represent a next generation of tissue engineered cardiovascular implants.

Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast.

PubMed

Kaplan, Tommy; Liu, Chih Long; Erkmann, Judith A; Holik, John; Grunstein, Michael; Kaufman, Paul D; Friedman, Nir; Rando, Oliver J

2008-11-01

Acetylation of histone H3 lysine 56 is a covalent modification best known as a mark of newly replicated chromatin, but it has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase and a further specific decrease in turnover at several early origins of replication, which switch from rapidly replaced in G1 phase to stably bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle and suggest that H3K56 acetylation provides a positive-feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.

Immunodetection of human topoisomerase I-DNA covalent complexes

PubMed Central

Patel, Anand G.; Flatten, Karen S.; Peterson, Kevin L.; Beito, Thomas G.; Schneider, Paula A.; Perkins, Angela L.; Harki, Daniel A.; Kaufmann, Scott H.

2016-01-01

A number of established and investigational anticancer drugs slow the religation step of DNA topoisomerase I (topo I). These agents induce cytotoxicity by stabilizing topo I-DNA covalent complexes, which in turn interact with advancing replication forks or transcription complexes to generate lethal lesions. Despite the importance of topo I-DNA covalent complexes, it has been difficult to detect these lesions within intact cells and tumors. Here, we report development of a monoclonal antibody that specifically recognizes covalent topo I-DNA complexes, but not free topo I or DNA, by immunoblotting, immunofluorescence or flow cytometry. Utilizing this antibody, we demonstrate readily detectable topo I-DNA covalent complexes after treatment with camptothecins, indenoisoquinolines and cisplatin but not nucleoside analogues. Topotecan-induced topo I-DNA complexes peak at 15–30 min after drug addition and then decrease, whereas indotecan-induced complexes persist for at least 4 h. Interestingly, simultaneous staining for covalent topo I-DNA complexes, phospho-H2AX and Rad51 suggests that topotecan-induced DNA double-strand breaks occur at sites distinct from stabilized topo I-DNA covalent complexes. These studies not only provide new insight into the action of topo I-directed agents, but also illustrate a strategy that can be applied to study additional topoisomerases and their inhibitors in vitro and in vivo. PMID:26917015

Single-Nucleosome Mapping of Histone Modifications in S. cerevisiae

PubMed Central

Kim, Minkyu; Buratowski, Stephen; Schreiber, Stuart L; Friedman, Nir

2005-01-01

Covalent modification of histone proteins plays a role in virtually every process on eukaryotic DNA, from transcription to DNA repair. Many different residues can be covalently modified, and it has been suggested that these modifications occur in a great number of independent, meaningful combinations. Published low-resolution microarray studies on the combinatorial complexity of histone modification patterns suffer from confounding effects caused by the averaging of modification levels over multiple nucleosomes. To overcome this problem, we used a high-resolution tiled microarray with single-nucleosome resolution to investigate the occurrence of combinations of 12 histone modifications on thousands of nucleosomes in actively growing S. cerevisiae. We found that histone modifications do not occur independently; there are roughly two groups of co-occurring modifications. One group of lysine acetylations shows a sharply defined domain of two hypo-acetylated nucleosomes, adjacent to the transcriptional start site, whose occurrence does not correlate with transcription levels. The other group consists of modifications occurring in gradients through the coding regions of genes in a pattern associated with transcription. We found no evidence for a deterministic code of many discrete states, but instead we saw blended, continuous patterns that distinguish nucleosomes at one location (e.g., promoter nucleosomes) from those at another location (e.g., over the 3′ ends of coding regions). These results are consistent with the idea of a simple, redundant histone code, in which multiple modifications share the same role. PMID:16122352

Covalently coupled hybrid of graphitic carbon nitride with reduced graphene oxide as a superior performance lithium-ion battery anode

NASA Astrophysics Data System (ADS)

Fu, Yongsheng; Zhu, Junwu; Hu, Chong; Wu, Xiaodong; Wang, Xin

2014-10-01

An in situ chemical synthetic approach has been designed for the fabrication of a covalently coupled hybrid consisting of graphitic carbon nitride (g-C3N4) with reduced graphene oxide (rGO) with differing g-C3N4/rGO ratio. The epoxy groups of graphene oxide (GO) undergo a nucleophilic substitution reaction with dicyandiamide (C2H4N4) to form the C2H4N4-GO composite via a covalent C-N bond, and then both the in situ polymerization of C2H4N4 and the thermal reduction of GO can be achieved at higher temperatures, forming the covalently coupled g-C3N4-rGO. FT-IR, CP-MAS NMR and XPS analyses, clearly revealed a covalent interaction between the g-C3N4 and rGO sheets. The g-C3N4-rGO exhibits an unprecedented high, stable and reversible capacity of 1525 mA h g-1 at a current density of 100 mA g-1 after 50 cycles. Even at a large current density of 1000 mA g-1, a reversible capacity of 943 mA h g-1 can still be retained. The superior electrochemical performance of g-C3N4-rGO is attributed to the specific characteristics of the unique nanostructure of g-C3N4-rGO and the concerted effects of g-C3N4 and rGO, including covalent interactions between the two moieties, the good conductivity and high special surface area of the nanocomposite, as well as the template effect of the planar amino group of g-C3N4 for the dispersed decoration of Li+ ions.An in situ chemical synthetic approach has been designed for the fabrication of a covalently coupled hybrid consisting of graphitic carbon nitride (g-C3N4) with reduced graphene oxide (rGO) with differing g-C3N4/rGO ratio. The epoxy groups of graphene oxide (GO) undergo a nucleophilic substitution reaction with dicyandiamide (C2H4N4) to form the C2H4N4-GO composite via a covalent C-N bond, and then both the in situ polymerization of C2H4N4 and the thermal reduction of GO can be achieved at higher temperatures, forming the covalently coupled g-C3N4-rGO. FT-IR, CP-MAS NMR and XPS analyses, clearly revealed a covalent interaction between

Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

NASA Technical Reports Server (NTRS)

Weber, Arthur L.

1989-01-01

Glyceraldehyde-3-phosphate acts as the substrate in a model of early self-replication of a phosphodiester copolymer of glycerate-3-phosphate and glycerol-3-phosphate. This model of self-replication is based on covalent complementarity in which information transfer is mediated by a single covalent bond, in contrast to multiple weak interactions that establish complementarity in nucleic acid replication. This replication model is connected to contemporary biochemistry through its use of glyceraldehyde-3-phosphate, a central metabolite of glycolysis and photosynthesis.

Comparative Mg(2+)-dependent sequential covalent binding stoichiometries of 3'-O-(4-benzoyl)benzoyl adenosine 5'-diphosphate of MF1, TF1, and the alpha 3 beta 3 core complex of TF1. The binding change motif is independent of the F1 gamma delta epsilon subunits.

PubMed

Aloise, P; Kagawa, Y; Coleman, P S

1991-06-05

Three F1 preparations, the beef heart (MF1) and thermophilic bacterium (TF1) holoenzymes, and the alpha 3 beta 3 "core" complex of TF1 reconstituted from individually expressed alpha and beta subunits, were compared as to their kinetic and binding stoichiometric responses to covalent photoaffinity labeling with BzATP and BzADP (+/- Mg2+). Each enzyme displayed an enhanced pseudo-first order rate of photoinhibition and one-third of the sites covalent binding to a catalytic site for full inhibition, plus, but not minus Mg2+. Titration of near stoichiometric [MgBzADP]/[F1] ratios during photolysis disclosed two sequential covalent binding patterns for each enzyme; a high affinity binding corresponding to unistoichiometric covalent association concomitant with enzyme inhibition, followed by a low affinity multisite-saturating covalent association. Thus, in the absence of the structural asymmetry inducing gamma delta epsilon subunits of the holoenzyme, the sequential binding of nucleotide at putative catalytic sites on the alpha 3 beta 3 complex of any F1 appears sufficient to effect binding affinity changes. With MF1, final covalent saturation of BzADP-accessible sites was achieved with 2 mol of BzADP/mol of enzyme, but with TF1 or its alpha 3 beta 3 complex, saturation required 3 mol of BzADP/mol of enzyme. Such differential final labeling stoichiometries could arise because of the endogenous presence of 1 nucleotide already bound to one of the 3 potential catalytic sites on normally prepared MF1, whereas TF1, possessing no endogenous nucleotide, has 3 vacant BzADP-accessible sites. Kinetics measurements revealed that regardless of the incremental extent of inhibition of the TF1 holoenzyme by BzADP during photolysis, the two higher apparent Km values (approximately 1.5 x 10(-4) and approximately 10(-3) M, respectively) of the progressively inactivated incubation are unchanged relative to fully unmodified enzyme. As reported for BzATP (or BzADP) and MF1 (Ackerman, S.H

Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains.

PubMed

de la Peña, Pilar; Domínguez, Pedro; Barros, Francisco

2018-03-01

Kv11.1 (hERG, KCNH2) is a voltage-gated potassium channel crucial in setting the cardiac rhythm and the electrical behaviour of several non-cardiac cell types. Voltage-dependent gating of Kv11.1 can be reconstructed from non-covalently linked voltage sensing and pore modules (split channels), challenging classical views of voltage-dependent channel activation based on a S4-S5 linker acting as a rigid mechanical lever to open the gate. Progressive displacement of the split position from the end to the beginning of the S4-S5 linker induces an increasing negative shift in activation voltage dependence, a reduced z g value and a more negative ΔG 0 for current activation, an almost complete abolition of the activation time course sigmoid shape and a slowing of the voltage-dependent deactivation. Channels disconnected at the S4-S5 linker near the S4 helix show a destabilization of the closed state(s). Furthermore, the isochronal ion current mode shift magnitude is clearly reduced in the different splits. Interestingly, the progressive modifications of voltage dependence activation gating by changing the split position are accompanied by a shift in the voltage-dependent availability to a methanethiosulfonate reagent of a Cys introduced at the upper S4 helix. Our data demonstrate for the first time that alterations in the covalent connection between the voltage sensor and the pore domains impact on the structural reorganizations of the voltage sensor domain. Also, they support the hypothesis that the S4-S5 linker integrates signals coming from other cytoplasmic domains that constitute either an important component or a crucial regulator of the gating machinery in Kv11.1 and other KCNH channels.

Scaled MP3 non-covalent interaction energies agree closely with accurate CCSD(T) benchmark data.

PubMed

Pitonák, Michal; Neogrády, Pavel; Cerný, Jirí; Grimme, Stefan; Hobza, Pavel

2009-01-12

Scaled MP3 interaction energies calculated as a sum of MP2/CBS (complete basis set limit) interaction energies and scaled third-order energy contributions obtained in small or medium size basis sets agree very closely with the estimated CCSD(T)/CBS interaction energies for the 22 H-bonded, dispersion-controlled and mixed non-covalent complexes from the S22 data set. Performance of this so-called MP2.5 (third-order scaling factor of 0.5) method has also been tested for 33 nucleic acid base pairs and two stacked conformers of porphine dimer. In all the test cases, performance of the MP2.5 method was shown to be superior to the scaled spin-component MP2 based methods, e.g. SCS-MP2, SCSN-MP2 and SCS(MI)-MP2. In particular, a very balanced treatment of hydrogen-bonded compared to stacked complexes is achieved with MP2.5. The main advantage of the approach is that it employs only a single empirical parameter and is thus biased by two rigorously defined, asymptotically correct ab-initio methods, MP2 and MP3. The method is proposed as an accurate but computationally feasible alternative to CCSD(T) for the computation of the properties of various kinds of non-covalently bound systems.

Biofunctional paper via the covalent modification of cellulose.

PubMed

Yu, Arthur; Shang, Jing; Cheng, Fang; Paik, Bradford A; Kaplan, Justin M; Andrade, Rodrigo B; Ratner, Daniel M

2012-07-31

Paper-based analytical devices are the subject of growing interest for the development of low-cost point-of-care diagnostics, environmental monitoring technologies, and research tools for limited-resource settings. However, there are limited chemistries available for the conjugation of biomolecules to cellulose for use in biomedical applications. Herein, divinyl sulfone (DVS) chemistry was demonstrated to immobilize small molecules, proteins, and DNA covalently onto the hydroxyl groups of cellulose membranes through nucleophilic addition. Assays on modified cellulose using protein-carbohydrate and protein-glycoprotein interactions as well as oligonucleotide hybridization showed that the membrane's bioactivity was specific, dose-dependent, and stable over a long period of time. The use of an inkjet printer to form patterns of biomolecules on DVS-activated cellulose illustrates the adaptability of the DVS functionalization technique to pattern sophisticated designs, with potential applications in cellulose-based lateral flow devices.

Multifunctional Pt(II) Reagents: Covalent Modifications of Pt Complexes Enable Diverse Structural Variation and In-Cell Detection.

PubMed

White, Jonathan D; Haley, Michael M; DeRose, Victoria J

2016-01-19

To enhance the functionality of Pt-based reagents, several strategies have been developed that utilize Pt compounds modified with small, reactive handles. This Account encapsulates work done by us and other groups regarding the use of Pt(II) compounds with reactive handles for subsequent elaboration with fluorophores or other functional moieties. Described strategies include the incorporation of substituents for well-known condensation or nucleophilic displacement-type reactions and their use, for example, to tether spectroscopic handles to Pt reagents for in vivo investigation. Other chief uses of displacement-type reactions have included tethering various small molecules exhibiting pharmacological activity directly to Pt, thus adding synergistic effects. Click chemistry-based ligation techniques have also been applied, primarily with azide- and alkyne-appended Pt complexes. Orthogonally reactive click chemistry reactions have proven invaluable when more traditional nucleophilic displacement reactions induce side-reactivity with the Pt center or when systematic functionalization of a larger number of Pt complexes is desired. Additionally, a diverse assortment of Pt-fluorophore conjugates have been tethered via click chemistry conjugation. In addition to providing a convenient synthetic path for diversifying Pt compounds, the use of click-capable Pt complexes has proved a powerful strategy for postbinding covalent modification and detection with fluorescent probes. This strategy bypasses undesirable influences of the fluorophore camouflaged as reactivity due to Pt that may be present when detecting preattached Pt-fluorophore conjugates. Using postbinding strategies, Pt reagent distributions in HeLa and lung carcinoma (NCI-H460) cell cultures were observed with two different azide-modified Pt compounds, a monofunctional Pt(II)-acridine type and a difunctional Pt(II)-neutral complex. In addition, cellular distribution was observed with an alkyne-appended difunctional

Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone.

PubMed

Hurd, P J; Whitmarsh, A J; Baldwin, G S; Kelly, S M; Waltho, J P; Price, N C; Connolly, B A; Hornby, D P

1999-02-19

DNA duplexes in which the target cytosine base is replaced by 2-H pyrimidinone have previously been shown to bind with a significantly greater affinity to C5-cytosine DNA methyltransferases than unmodified DNA. Here, it is shown that 2-H pyrimidinone, when incorporated into DNA duplexes containing the recognition sites for M.HgaI-2 and M.MspI, elicits the formation of inhibitory covalent nucleoprotein complexes. We have found that although covalent complexes are formed between 2-H pyrimidinone-modified DNA and both M.HgaI-2 and M.MspI, the kinetics of complex formation are quite distinct in each case. Moreover, the formation of a covalent complex is still observed between 2-H pyrimidinone DNA and M.MspI in which the active-site cysteine residue is replaced by serine or threonine. Covalent complex formation between M.MspI and 2-H pyrimidinone occurs as a direct result of nucleophilic attack by the residue at the catalytic position, which is enhanced by the absence of the 4-amino function in the base. The substitution of the catalytic cysteine residue by tyrosine or chemical modification of the wild-type enzyme with N-ethylmaleimide, abolishes covalent interaction. Nevertheless the 2-H pyrimidinone-substituted duplex still binds to M.MspI with a greater affinity than a standard cognate duplex, since the 2-H pyrimidinone base is mis-paired with guanine. Copyright 1999 Academic Press.

pH-Responsive Nanoscale Covalent Organic Polymers as a Biodegradable Drug Carrier for Combined Photodynamic Chemotherapy of Cancer.

PubMed

Wang, Hairong; Zhu, Wenwen; Liu, Jingjing; Dong, Ziliang; Liu, Zhuang

2018-05-02

Covalent organic polymers (COPs) are a promising class of cross-linked polymeric networks and porous structures composed of covalent organic molecules that attract extensive attention. Despite increasing interest in applying COPs for applications in nanomedicine, the pH-sensitive COPs that are able to sensitively respond to the slightly acidic tumor microenvironment for tumor-specific drug delivery and therapy remain to be explored to our best knowledge. Herein, a new style of pH-responsive COPs were prepared using acryloyl meso-tetra( p-hydroxyphenyl) porphine (acryloyl-THPP) to react with 4,4'-trimethylene dipiperidine to form the pH-responsive cross-linked biodegradable β-amino esters (BAEs). Amine-modified poly(ethylene glycol) (PEG) was then introduced to terminate the reaction and form the PEG shell. The formulated pH-responsive THPP-BAE-PEG COPs can be utilized to encapsulate anticancer drug doxorubicin (DOX) due to their porous structure. Upon intravenous injection, such DOX-loaded COPs show a prolonged blood circulation as well as an efficient tumor accumulation. Along with the pH-triggered drug release for chemotherapy, the singlet oxygen produced by THPP under light exposure for photodynamic therapy would further endow us a combined treatment strategy, which offers synergistic antitumor effects in our in vivo tumor model experiments. Our study illustrates that COPs fabricated with tumor microenvironment responsive linkers may be a promising type of materials for applications in cancer nanomedicine.

Structural and Functional Modifications of Corneal Crystallin ALDH3A1 by UVB Light

PubMed Central

Estey, Tia; Chen, Ying; Carpenter, John F.; Vasiliou, Vasilis

2010-01-01

As one of the most abundantly expressed proteins in the mammalian corneal epithelium, aldehyde dehydrogenase 3A1 (ALDH3A1) plays critical and multifaceted roles in protecting the cornea from oxidative stress. Recent studies have demonstrated that one protective mechanism of ALDH3A1 is the direct absorption of UV-energy, which reduces damage to other corneal proteins such as glucose-6-phosphate dehydrogenase through a competition mechanism. UV-exposure, however, leads to the inactivation of ALDH3A1 in such cases. In the current study, we demonstrate that UV-light caused soluble, non-native aggregation of ALDH3A1 due to both covalent and non-covalent interactions, and that the formation of the aggregates was responsible for the loss of ALDH3A1 enzymatic activity. Spectroscopic studies revealed that as a result of aggregation, the secondary and tertiary structure of ALDH3A1 were perturbed. LysC peptide mapping using MALDI-TOF mass spectrometry shows that UV-induced damage to ALDH3A1 also includes chemical modifications to Trp, Met, and Cys residues. Surprisingly, the conserved active site Cys of ALDH3A1 does not appear to be affected by UV-exposure; this residue remained intact after exposure to UV-light that rendered the enzyme completely inactive. Collectively, our data suggest that the UV-induced inactivation of ALDH3A1 is a result of non-native aggregation and associated structural changes rather than specific damage to the active site Cys. PMID:21203538

Citrullination/Methylation Crosstalk on Histone H3 Regulates ER-Target Gene Transcription.

PubMed

Clancy, Kathleen W; Russell, Anna-Maria; Subramanian, Venkataraman; Nguyen, Hannah; Qian, Yuewei; Campbell, Robert M; Thompson, Paul R

2017-06-16

Posttranslational modifications of histone tails are a key contributor to epigenetic regulation. Histone H3 Arg26 and Lys27 are both modified by multiple enzymes, and their modifications have profound effects on gene expression. Citrullination of H3R26 by PAD2 and methylation of H3K27 by PRC2 have opposing downstream impacts on gene regulation; H3R26 citrullination activates gene expression, and H3K27 methylation represses gene expression. Both of these modifications are drivers of a variety of cancers, and their writer enzymes, PAD2 and EZH2, are the targets of drug therapies. After biochemical and cell-based analysis of these modifications, a negative crosstalk interaction is observed. Methylation of H3K27 slows citrullination of H3R26 30-fold, whereas citrullination of H3R26 slows methylation 30,000-fold. Examination of the mechanism of this crosstalk interaction uncovered a change in structure of the histone tail upon citrullination which prevents methylation by the PRC2 complex. This mechanism of crosstalk is reiterated in cell lines using knockdowns and inhibitors of both enzymes. Based our data, we propose a model in which, after H3 Cit26 formation, H3K27 demethylases are recruited to the chromatin to activate transcription. In total, our studies support the existence of crosstalk between citrullination of H3R26 and methylation of H3K27.

Advances in covalent organic frameworks in separation science.

PubMed

Qian, Hai-Long; Yang, Cheng-Xiong; Wang, Wen-Long; Yang, Cheng; Yan, Xiu-Ping

2018-03-23

Covalent organic frameworks (COFs) are a new class of multifunctional crystalline organic polymer constructed with organic monomers via robust covalent bonds. The unique properties such as convenient modification, low densities, large specific surface areas, good stability and permanent porosity make COFs great potential in separation science. This review shows the state-of-the art for the application of COFs and their composites in analytical separation science. COFs and their composites have been explored as promising sorbents for solid phase extraction, potential coatings for solid phase microextraction, and novel stationary phases for gas chromatography, high-performance liquid chromatography and capillary electrochromatography. The prospects of COFs for separation science are also presented, which can offer an outlook and reference for further study on the applications of COFs. Copyright © 2018 Elsevier B.V. All rights reserved.

Nanoengineered Ionic-Covalent Entanglement (NICE) Bioinks for 3D Bioprinting.

PubMed

Chimene, David; Peak, Charles W; Gentry, James L; Carrow, James K; Cross, Lauren M; Mondragon, Eli; Cardoso, Guinea B; Kaunas, Roland; Gaharwar, Akhilesh K

2018-03-28

We introduce an enhanced nanoengineered ionic-covalent entanglement (NICE) bioink for the fabrication of mechanically stiff and elastomeric 3D biostructures. NICE bioink formulations combine nanocomposite and ionic-covalent entanglement (ICE) strengthening mechanisms to print customizable cell-laden constructs for tissue engineering with high structural fidelity and mechanical stiffness. Nanocomposite and ICE strengthening mechanisms complement each other through synergistic interactions, improving mechanical strength, elasticity, toughness, and flow properties beyond the sum of the effects of either reinforcement technique alone. Herschel-Bulkley flow behavior shields encapsulated cells from excessive shear stresses during extrusion. The encapsulated cells readily proliferate and maintain high cell viability over 120 days within the 3D-printed structure, which is vital for long-term tissue regeneration. A unique aspect of the NICE bioink is its ability to print much taller structures, with higher aspect ratios, than can be achieved with conventional bioinks without requiring secondary supports. We envision that NICE bioinks can be used to bioprint complex, large-scale, cell-laden constructs for tissue engineering with high structural fidelity and mechanical stiffness for applications in custom bioprinted scaffolds and tissue engineered implants.

Chemical Bonding of AlH3 Hydride by Al-L2,3 Electron Energy-Loss Spectra and First-Principles Calculations

PubMed Central

Tatsumi, Kazuyoshi; Muto, Shunsuke; Ikeda, Kazutaka; Orimo, Shin-Ichi

2012-01-01

In a previous study, we used transmission electron microscopy and electron energy-loss (EEL) spectroscopy to investigate dehydrogenation of AlH3 particles. In the present study, we systematically examine differences in the chemical bonding states of Al-containing compounds (including AlH3) by comparing their Al-L2,3 EEL spectra. The spectral chemical shift and the fine peak structure of the spectra were consistent with the degree of covalent bonding of Al. This finding will be useful for future nanoscale analysis of AlH3 dehydrogenation toward the cell. PMID:28816996

H3K27 methylation and H3S28 phosphorylation-dependent transcriptional regulation by INHAT subunit SET/TAF-Iβ.

PubMed

Kim, Ji-Young; Kim, Kee-Beom; Son, Hye-Ju; Chae, Yun-Cheol; Oh, Si-Taek; Kim, Dong-Wook; Pak, Jhang Ho; Seo, Sang-Beom

2012-09-21

Significant progress has been made in understanding the relationship between histone modifications and 'reader' molecules and their effects on transcriptional regulation. A previously identified INHAT complex subunit, SET/TAF-Iβ, binds to histones and inhibits histone acetylation. To investigate the binding specificities of SET/TAF-Iβ to various histone modifications, we employed modified histone tail peptide array analyses. SET/TAF-Iβ strongly recognized PRC2-mediated H3K27me1/2/3; however, the bindings were completely disrupted by H3S28 phosphorylation. We have demonstrated that SET/TAF-Iβ is sequentially recruited to the target gene promoter ATF3 after the PRC2 complex via H3K27me recognition and may offer additive effects in the repression of the target gene. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Excitation Localization/Delocalization Isomerism in a Strongly Coupled Covalent Dimer of 1,3-Diphenylisobenzofuran

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schrauben, Joel N.; Akdag, Akin; Wen, Jin

Two isomers of both the lowest excited singlet (S1) and triplet (T1) states of the directly para, para'-connected covalent dimer of the singlet-fission chromophore 1,3-diphenylisobenzofuran have been observed. In one isomer, excitation is delocalized over both halves of the dimer, and in the other, it is localized on one or the other half. For a covalent dimer in solution, such 'excitation isomerism' is extremely rare. The vibrationally relaxed isomers do not interconvert, and their photophysical properties, including singlet fission, differ significantly.

Core-crosslinked polymeric micelles with controlled release of covalently entrapped doxorubicin.

PubMed

Talelli, Marina; Iman, Maryam; Varkouhi, Amir K; Rijcken, Cristianne J F; Schiffelers, Raymond M; Etrych, Tomas; Ulbrich, Karel; van Nostrum, Cornelus F; Lammers, Twan; Storm, Gert; Hennink, Wim E

2010-10-01

Doxorubicin (DOX) is clinically applied in cancer therapy, but its use is associated with dose limiting severe side effects. Core-crosslinked biodegradable polymeric micelles composed of poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p(HPMAm-Lac(n))) diblock copolymers have shown prolonged circulation in the blood stream upon intravenous administration and enhanced tumor accumulation through the enhanced permeation and retention (EPR) effect. However a (physically) entrapped anticancer drug (paclitaxel) was previously shown to be rapidly eliminated from the circulation, likely because the drug was insufficiently retained in the micelles. To fully exploit the EPR effect for drug targeting, a DOX methacrylamide derivative (DOX-MA) was covalently incorporated into the micellar core by free radical polymerization. The structure of the doxorubicin derivative is susceptible to pH-sensitive hydrolysis, enabling controlled release of the drug in acidic conditions (in either the intratumoral environment and/or the endosomal vesicles). 30-40% w/w of the added drug was covalently entrapped, and the micelles with covalently entrapped DOX had an average diameter of 80 nm. The entire drug payload was released within 24 h incubation at pH 5 and 37 degrees C, whereas only around 5% release was observed at pH 7.4. DOX micelles showed higher cytotoxicity in B16F10 and OVCAR-3 cells compared to DOX-MA, likely due to cellular uptake of the micelles via endocytosis and intracellular drug release in the acidic organelles. The micelles showed better anti-tumor activity than free DOX in mice bearing B16F10 melanoma carcinoma. The results presented in this paper show that mPEG-b-p(HPMAm-Lac(n)) polymeric micelles with covalently entrapped doxorubicin is a system highly promising for the targeted delivery of cytostatic agents. Copyright 2010 Elsevier Ltd. All rights reserved.

Communication: Many-body stabilization of non-covalent interactions: Structure, stability, and mechanics of Ag3Co(CN)6 framework.

PubMed

Liu, Xiaofei; Hermann, Jan; Tkatchenko, Alexandre

2016-12-28

Stimuli-responsive metal-organic frameworks (MOFs) and other framework materials exhibit a broad variety of useful properties, which mainly stem from an interplay of strong covalent bonds within the organic linkers with presumably weak van der Waals (vdW) interactions which determine the overall packing of the framework constituents. Using Ag 3 Co(CN) 6 as a fundamental test case-a system with a colossal positive and negative thermal expansion [A. L. Goodwin et al., Science 319, 794 (2008)]-we demonstrate that its structure, stability, dielectric, vibrational, and mechanical properties are critically influenced by many-body electronic correlation contributions to non-covalent vdW interactions. The Ag 3 Co(CN) 6 framework is a remarkable molecular crystal, being visibly stabilized, rather than destabilized, by many-body vdW correlations. A detailed comparison with H 3 Co(CN) 6 highlights the crucial role of strongly polarized metallophilic interactions in dictating the exceptional properties of denser MOFs. Beyond MOFs, our findings indicate that many-body electronic correlations can substantially stabilize polarizable materials, providing a novel mechanism for tuning the properties of nanomaterials with intricate structural motifs.

An efficient immunodetection method for histone modifications in plants.

PubMed

Nic-Can, Geovanny; Hernández-Castellano, Sara; Kú-González, Angela; Loyola-Vargas, Víctor M; De-la-Peña, Clelia

2013-12-16

Epigenetic mechanisms can be highly dynamic, but the cross-talk among them and with the genome is still poorly understood. Many of these mechanisms work at different places in the cell and at different times of organism development. Covalent histone modifications are one of the most complex and studied epigenetic mechanisms involved in cellular reprogramming and development in plants. Therefore, the knowledge of the spatial distribution of histone methylation in different tissues is important to understand their behavior on specific cells. Based on the importance of epigenetic marks for biology, we present a simplified, inexpensive and efficient protocol for in situ immunolocalization on different tissues such as flowers, buds, callus, somatic embryo and meristematic tissue from several plants of agronomical and biological importance. Here, we fully describe all the steps to perform the localization of histone modifications. Using this method, we were able to visualize the distribution of H3K4me3 and H3K9me2 without loss of histological integrity of tissues from several plants, including Agave tequilana, Capsicum chinense, Coffea canephora and Cedrela odorata, as well as Arabidopsis thaliana. There are many protocols to study chromatin modifications; however, most of them are expensive, difficult and require sophisticated equipment. Here, we provide an efficient protocol for in situ localization of histone methylation that dispenses with the use of expensive and sensitive enzymes. The present method can be used to investigate the cellular distribution and localization of a wide array of proteins, which could help to clarify the biological role that they play at specific times and places in different tissues of various plant species.

Covalent Organic Framework Functionalized with 8-Hydroxyquinoline as a Dual-Mode Fluorescent and Colorimetric pH Sensor.

PubMed

Chen, Long; He, Linwei; Ma, Fuyin; Liu, Wei; Wang, Yaxing; Silver, Mark A; Chen, Lanhua; Zhu, Lin; Gui, Daxiang; Diwu, Juan; Chai, Zhifang; Wang, Shuao

2018-05-09

Real-time and accurate detection of pH in aqueous solution is of great significance in chemical, environmental, and engineering-related fields. We report here the use of 8-hydroxyquinoline-functionalized covalent organic framework (COF-HQ) for dual-mode pH sensing. In the fluorescent mode, the emission intensity of COF-HQ weakened as the pH decreased, and also displayed a good linear relationship against pH in the range from 1 to 5. In addition, COF-HQ showed discernible color changes from yellow to black as the acidity increased and can be therefore used as a colorimetric pH sensor. All these changes are reversible and COF-HQ can be recycled for multiple detection runs owing to its high hydrolytical stability. It can be further assembled into a mixed matrix membrane for practical applications.

Enhancement of CO Evolution by Modification of Ga2O3 with Rare-Earth Elements for the Photocatalytic Conversion of CO2 by H2O.

PubMed

Tatsumi, Hiroyuki; Teramura, Kentaro; Huang, Zeai; Wang, Zheng; Asakura, Hiroyuki; Hosokawa, Saburo; Tanaka, Tsunehiro

2017-12-12

Modification of the surface of Ga 2 O 3 with rare-earth elements enhanced the evolution of CO as a reduction product in the photocatalytic conversion of CO 2 using H 2 O as an electron donor under UV irradiation in aqueous NaHCO 3 as a pH buffer, with the rare-earth species functioning as a CO 2 capture and storage material. Isotope experiments using 13 CO 2 as a substrate clearly revealed that CO was generated from the introduced gaseous CO 2 . In the presence of the NaHCO 3 additive, the rare-earth (RE) species on the Ga 2 O 3 surface are transformed into carbonate hydrates (RE 2 (CO 3 ) 3 ·nH 2 O) and/or hydroxycarbonates (RE 2 (OH) 2(3-x) (CO 3 ) x ) which are decomposed upon photoirradiation. Consequently, Ag-loaded Yb-modified Ga 2 O 3 exhibits much higher activity (209 μmol h -1 of CO) than the pristine Ag-loaded Ga 2 O 3 . The further modification of the surface of the Yb-modified Ga 2 O 3 with Zn afforded a selectivity toward CO evolution of 80%. Thus, we successfully achieved an efficient Ag-loaded Yb- and Zn-modified Ga 2 O 3 photocatalyst with high activity and controllable selectivity, suitable for use in artificial photosynthesis.

Oxadiazole-isopropylamides as Potent and Non-covalent Proteasome Inhibitors

PubMed Central

Ozcan, Sevil; Kazi, Aslamuzzaman; Marsilio, Frank; Fang, Bin; Guida, Wayne C.; Koomen, John; Lawrence, Harshani R.; Sebti, Saïd M.

2013-01-01

Screening of the 50,000 ChemBridge compound library led to the identification of the oxadiazole-isopropylamide 1 (PI-1833) which inhibited CT-L activity (IC50 0.60 μM) with little effects on the other 2 major proteasome proteolytic activities, T-L and PGPH-L. LC/MS-MS and dialysis show that 1 is a non-covalent and rapidly reversible CT-L inhibitor. Focused library synthesis provided 11ad (PI-1840) with CT-L activity (IC50 27 nM). Detailed SAR studies indicate that the amide moiety and the 2 phenyl rings are sensitive toward modifications. Hydrophobic residues, such as propyl or butyl, in the para-position (not ortho or meta) of the A-ring and a meta-pyridyl group as B-ring significantly improve activity. Compound 11ad (IC50 0.37 μM) is more potent than 1 (IC50 3.5 μM) at inhibiting CT-L activity in intact MDA-MB-468 human breast cancer cells and inhibiting their survival. The activity of 11ad warrants further pre-clinical investigation of this class as non-covalent proteasome inhibitors. PMID:23547706

CovalentDock Cloud: a web server for automated covalent docking.

PubMed

Ouyang, Xuchang; Zhou, Shuo; Ge, Zemei; Li, Runtao; Kwoh, Chee Keong

2013-07-01

Covalent binding is an important mechanism for many drugs to gain its function. We developed a computational algorithm to model this chemical event and extended it to a web server, the CovalentDock Cloud, to make it accessible directly online without any local installation and configuration. It provides a simple yet user-friendly web interface to perform covalent docking experiments and analysis online. The web server accepts the structures of both the ligand and the receptor uploaded by the user or retrieved from online databases with valid access id. It identifies the potential covalent binding patterns, carries out the covalent docking experiments and provides visualization of the result for user analysis. This web server is free and open to all users at http://docking.sce.ntu.edu.sg/.

CovalentDock Cloud: a web server for automated covalent docking

PubMed Central

Ouyang, Xuchang; Zhou, Shuo; Ge, Zemei; Li, Runtao; Kwoh, Chee Keong

2013-01-01

Covalent binding is an important mechanism for many drugs to gain its function. We developed a computational algorithm to model this chemical event and extended it to a web server, the CovalentDock Cloud, to make it accessible directly online without any local installation and configuration. It provides a simple yet user-friendly web interface to perform covalent docking experiments and analysis online. The web server accepts the structures of both the ligand and the receptor uploaded by the user or retrieved from online databases with valid access id. It identifies the potential covalent binding patterns, carries out the covalent docking experiments and provides visualization of the result for user analysis. This web server is free and open to all users at http://docking.sce.ntu.edu.sg/. PMID:23677616

Sulfonyl chlorides as an efficient tool for the postsynthetic modification of Cr-MIL-101-SO3H and CAU-1-NH2.

PubMed

Klinkebiel, Arne; Reimer, Nele; Lammert, Martin; Stock, Norbert; Lüning, Ulrich

2014-08-25

Postsynthetic modification can be used to introduce sulfonamide functionalities into MOF frameworks. Using sulfonyl chlorides as reactive intermediates, Cr-MIL-SO3H and CAU-1-NH2 have been further modified to give hitherto unknown functionalized MOFs in which a sulfonamide group is bound to the framework either by its N or its S atom.

Systems Level Analysis of Histone H3 Post-translational Modifications (PTMs) Reveals Features of PTM Crosstalk in Chromatin Regulation*

PubMed Central

Schwämmle, Veit; Sidoli, Simone; Ruminowicz, Chrystian; Wu, Xudong; Lee, Chung-Fan; Helin, Kristian; Jensen, Ole N.

2016-01-01

Histones are abundant chromatin constituents carrying numerous post-translational modifications (PTMs). Such PTMs mediate a variety of biological functions, including recruitment of enzymatic readers, writers and erasers that modulate DNA replication, transcription and repair. Individual histone molecules contain multiple coexisting PTMs, some of which exhibit crosstalk, i.e. coordinated or mutually exclusive activities. Here, we present an integrated experimental and computational systems level molecular characterization of histone PTMs and PTM crosstalk. Using wild type and engineered mouse embryonic stem cells (mESCs) knocked out in components of the Polycomb Repressive Complex 2 (PRC2, Suz12−/−), PRC1 (Ring1A/B−/−) and (Dnmt1/3a/3b−/−) we performed comprehensive PTM analysis of histone H3 tails (50 aa) by utilizing quantitative middle-down proteome analysis by tandem mass spectrometry. We characterized combinatorial PTM features across the four mESC lines and then applied statistical data analysis to predict crosstalk between histone H3 PTMs. We detected an overrepresentation of positive crosstalk (codependent marks) between adjacent mono-methylated and acetylated marks, and negative crosstalk (mutually exclusive marks) among most of the seven characterized di- and tri-methylated lysine residues in the H3 tails. We report novel features of PTM interplay involving hitherto poorly characterized arginine methylation and lysine methylation sites, including H3R2me, H3R8me and H3K37me. Integration of the H3 data with RNAseq data by coabundance clustering analysis of histone PTMs and histone modifying enzymes revealed correlations between PTM and enzyme levels. We conclude that middle-down proteomics is a powerful tool to determine conserved or dynamic interdependencies between histone marks, which paves the way for detailed investigations of the histone code. Histone H3 PTM data is publicly available in the CrossTalkDB repository at http

Covalent and non-covalent chemical engineering of actin for biotechnological applications.

PubMed

Kumar, Saroj; Mansson, Alf

2017-11-15

The cytoskeletal filaments are self-assembled protein polymers with 8-25nm diameters and up to several tens of micrometres length. They have a range of pivotal roles in eukaryotic cells, including transportation of intracellular cargoes (primarily microtubules with dynein and kinesin motors) and cell motility (primarily actin and myosin) where muscle contraction is one example. For two decades, the cytoskeletal filaments and their associated motor systems have been explored for nanotechnological applications including miniaturized sensor systems and lab-on-a-chip devices. Several developments have also revolved around possible exploitation of the filaments alone without their motor partners. Efforts to use the cytoskeletal filaments for applications often require chemical or genetic engineering of the filaments such as specific conjugation with fluorophores, antibodies, oligonucleotides or various macromolecular complexes e.g. nanoparticles. Similar conjugation methods are also instrumental for a range of fundamental biophysical studies. Here we review methods for non-covalent and covalent chemical modifications of actin filaments with focus on critical advantages and challenges of different methods as well as critical steps in the conjugation procedures. We also review potential uses of the engineered actin filaments in nanotechnological applications and in some key fundamental studies of actin and myosin function. Finally, we consider possible future lines of investigation that may be addressed by applying chemical conjugation of actin in new ways. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Molecular Biodynamers: Dynamic Covalent Analogues of Biopolymers

PubMed Central

2017-01-01

Conspectus Constitutional dynamic chemistry (CDC) features the use of reversible linkages at both molecular and supramolecular levels, including reversible covalent bonds (dynamic covalent chemistry, DCC) and noncovalent interactions (dynamic noncovalent chemistry, DNCC). Due to its inherent reversibility and stimuli-responsiveness, CDC has been widely utilized as a powerful tool for the screening of bioactive compounds, the exploitation of receptors or substrates driven by molecular recognition, and the fabrication of constitutionally dynamic materials. Implementation of CDC in biopolymer science leads to the generation of constitutionally dynamic analogues of biopolymers, biodynamers, at the molecular level (molecular biodynamers) through DCC or at the supramolecular level (supramolecular biodynamers) via DNCC. Therefore, biodynamers are prepared by reversible covalent polymerization or noncovalent polyassociation of biorelevant monomers. In particular, molecular biodynamers, biodynamers of the covalent type whose monomeric units are connected by reversible covalent bonds, are generated by reversible polymerization of bio-based monomers and can be seen as a combination of biopolymers with DCC. Owing to the reversible covalent bonds used in DCC, molecular biodynamers can undergo continuous and spontaneous constitutional modifications via incorporation/decorporation and exchange of biorelevant monomers in response to internal or external stimuli. As a result, they behave as adaptive materials with novel properties, such as self-healing, stimuli-responsiveness, and tunable mechanical and optical character. More specifically, molecular biodynamers combine the biorelevant characters (e.g., biocompatibility, biodegradability, biofunctionality) of bioactive monomers with the dynamic features of reversible covalent bonds (e.g., changeable, tunable, controllable, self-healing, and stimuli-responsive capacities), to realize synergistic properties in one system. In addition

Microenvironmental pH-modification to improve dissolution behavior and oral absorption for drugs with pH-dependent solubility.

PubMed

Taniguchi, Chika; Kawabata, Yohei; Wada, Koichi; Yamada, Shizuo; Onoue, Satomi

2014-04-01

Drug release and oral absorption of drugs with pH-dependent solubility are influenced by the conditions in the gastrointestinal tract. In some cases, poor oral absorption has been observed for these drugs, causing insufficient drug efficacy. The pH-modification of a formulation could be a promising approach to overcome the poor oral absorption of drugs with pH-dependent solubility. The present review aims to summarize the pH-modifier approach and strategic analyses of microenvironmental pH for formulation design and development. We also provide literature- and patent-based examples of the application of pH-modification technology to solid dosage forms. For the pH-modification approach, the microenvironmental pH at the diffusion area can be altered by dissolving pH-modifying excipients in the formulation. The modulation of the microenvironmental pH could improve dissolution behavior of drugs with pH-dependent solubility, possibly leading to better oral absorption. According to this concept, the modulated level of microenvironmental pH and its duration can be key factors for improvement in drug dissolution. The measurement of microenvironmental pH and release of pH-modifier would provide theoretical insight for the selection of an appropriate pH-modifier and optimization of the formulation.

Photochemical properties and sensor applications of modified yellow fluorescent protein (YFP) covalently attached to the surfaces of etched optical fibers (EOFs).

PubMed

Veselov, Alexey A; Abraham, Bobin George; Lemmetyinen, Helge; Karp, Matti T; Tkachenko, Nikolai V

2012-01-01

Fluorescent proteins have the inherent ability to act as sensing components which function both in vitro and inside living cells. We describe here a novel study on a covalent site-specific bonding of fluorescent proteins to form self-assembled monolayers (SAMs) on the surface of etched optical fibers (EOFs). Deposition of fluorescent proteins on EOFs gives the opportunity to increase the interaction of guided light with deposited molecules relative to plane glass surfaces. The EOF modification is carried out by surface activation using 3-aminopropylthrimethoxysilane (APTMS) and bifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) which exposes sulfhydryl-reactive maleimide groups followed by covalent site-specific coupling of modified yellow fluorescent protein (YFP). Steady-state and fluorescence lifetime measurements confirm the formation of SAM. The sensor applications of YPF SAMs on EOF are demonstrated by the gradual increase of emission intensity upon addition of Ca(2+) ions in the concentration range from a few tens of micromolars up to a few tens of millimolars. The studies on the effect of pH, divalent cations, denaturing agents, and proteases reveal the stability of YFP on EOFs at normal physiological conditions. However, treatments with 0.5% SDS at pH 8.5 and protease trypsin are found to denaturate or cleave the YFP from fiber surfaces.

Uranus' (3-0) H2 quadrupole line profiles

NASA Technical Reports Server (NTRS)

Trafton, L.

1987-01-01

Spectra of Uranus' S3(0) and S3(1) H2 quadrupole lines, obtained during the 1978-1980 apparitions, are analyzed, and are found to require the presence of a deep cloud. Modifications of the Baines and Bergstralh (1986) standard model, including an additional haze layer above the 16-km-am H2 level which contains strongly absorbing particles, are needed to fit the observations. For a Rayleigh phase function, such a haze (uniformly mixed with the gas above this level) would have an absorption optical depth of 0.16 and a single scattering particle albedo of 0.30. This modification would imply a fraction of normal H2 equal to 0.25 + or - 0.10, in agreement with the Baines and Bergstralh standard model.

Covalent functionalized black phosphorus quantum dots

NASA Astrophysics Data System (ADS)

Scotognella, Francesco; Kriegel, Ilka; Sassolini, Simone

2018-01-01

Black phosphorus (BP) nanostructures enable a new strategy to tune the electronic and optical properties of this atomically thin material. In this paper we show, via density functional theory calculations, the possibility to modify the optical properties of BP quantum dots via covalent functionalization. The quantum dot selected in this study has chemical formula P24H12 and has been covalent functionalized with one or more benzene rings or anthracene. The effect of functionalization is highlighted in the absorption spectra, where a red shift of the absorption is noticeable. The shift can be ascribed to an electron delocalization in the black phosphorus/organic molecule nanostructure.

Facile fabrication of highly controllable gating systems based on the combination of inverse opal structure and dynamic covalent chemistry.

PubMed

Wang, Chen; Yang, Haowei; Tian, Li; Wang, Shiqiang; Gao, Ning; Zhang, Wanlin; Wang, Peng; Yin, Xianpeng; Li, Guangtao

2017-06-01

A three-dimensional (3D) inverse opal with periodic and porous structures has shown great potential for applications not only in optics and optoelectronics, but also in functional membranes. In this work, the benzaldehyde group was initially introduced into a 3D nanoporous inverse opal, serving as a platform for fabricating functional membranes. By employing the dynamic covalent approach, a highly controllable gating system was facilely fabricated to achieve modulable and reversible transport features. It was found that the physical/chemical properties and pore size of the gating system could easily be regulated through post-modification with amines. As a demonstration, the gated nanopores were modified with three kinds of amines to control the wettability, surface charge and nanopore size which in turn was exploited to achieve selective mass transport, including hydrophobic molecules, cations and anions, and the transport with respect to the physical steric hindrance. In particular, the gating system showed extraordinary reversibility and could recover to its pristine state by simply changing pH values. Due to the unlimited variety provided by the Schiff base reaction, the inverse opal described here exhibits a significant extendibility and could be easily post-modified with stimuli-responsive molecules for special purposes. Furthermore, this work can be extended to employ other dynamic covalent routes, for example Diels-Alder, ester exchange and disulfide exchange-based routes.

Biophysically defined and cytocompatible covalently adaptable networks as viscoelastic 3D cell culture systems.

PubMed

McKinnon, Daniel D; Domaille, Dylan W; Cha, Jennifer N; Anseth, Kristi S

2014-02-12

Presented here is a cytocompatible covalently adaptable hydrogel uniquely capable of mimicking the complex biophysical properties of native tissue and enabling natural cell functions without matrix degradation. Demonstrated is both the ability to control elastic modulus and stress relaxation time constants by more than an order of magnitude while predicting these values based on fundamental theoretical understanding and the simulation of muscle tissue and the encapsulation of myoblasts. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The covalent interaction between dihydrogen and gold: A rotational spectroscopic study of H2-AuCl

NASA Astrophysics Data System (ADS)

Obenchain, Daniel A.; Frank, Derek S.; Grubbs, G. S.; Pickett, Herbert M.; Novick, Stewart E.

2017-05-01

The pure rotational transitions of H2-AuCl have been measured using a pulsed-jet cavity Fourier transform microwave spectrometer equipped with a laser ablation source. The structure was found to be T-shaped, with the H-H bond interacting with the gold atom. Both 35Cl and 37Cl isotopologues have been measured for both ortho and para states of H2. Rotational constants, quartic centrifugal distortion constants, and nuclear quadrupole coupling constants for gold and chlorine have been determined. The use of the nuclear spin-nuclear spin interaction terms Daa, Dbb, and Dcc for H2 were required to fit the ortho state of hydrogen, as well as a nuclear-spin rotation constant Caa. The values of the nuclear quadrupole coupling constant of gold are χa a=-817.9929 (35 ) MHz, χb b=504.0 (27 ) MHz, and χc c=314.0 (27 ) . This is large compared to the eQq of AuCl, 9.63 312(13) MHz, which indicates a strong, covalent interaction between gold and dihydrogen.

Prodrug and covalent linker strategies for the solubilization of dual-action antioxidants/iron chelators.

PubMed

Bebbington, David; Dawson, Claire E; Gaur, Suneel; Spencer, John

2002-11-18

Water soluble prodrugs of hybrid free radical scavenger/iron chelating molecules, based on 3,5-disubstituted-4-hydroxyphenyl derivatives and 3-hydroxy-2-methyl-4(1H)-pyridinone (deferiprone), have been prepared. Related hybrid molecules containing a covalent poly(ethylene)glycol or an amine linker were also synthesized.

Capillary electrophoresis of covalently functionalized single-chirality carbon nanotubes.

PubMed

He, Pingli; Meany, Brendan; Wang, Chunyan; Piao, Yanmei; Kwon, Hyejin; Deng, Shunliu; Wang, YuHuang

2017-07-01

We demonstrate the separation of chirality-enriched single-walled carbon nanotubes (SWCNTs) by degree of surface functionalization using high-performance CE. Controlled amounts of negatively charged and positively charged functional groups were attached to the sidewall of chirality-enriched SWCNTs through covalent functionalization using 4-carboxybenzenediazonium tetrafluoroborate or 4-diazo-N,N-diethylaniline tetrafluoroborate, respectively. Surfactant- and pH-dependent studies confirmed that under conditions that minimized ionic screening effects, separation of these functionalized SWCNTs was strongly dependent on the surface charge density introduced through covalent surface chemistry. For both heterogeneous mixtures and single-chirality-enriched samples, covalently functionalized SWCNTs showed substantially increased peak width in electropherogram spectra compared to nonfunctionalized SWCNTs, which can be attributed to a distribution of surface charges along the functionalized nanotubes. Successful separation of functionalized single-chirality SWCNTs by functional density was confirmed with UV-Vis-NIR absorption and Raman scattering spectroscopies of fraction collected samples. These results suggest a high degree of structural heterogeneity in covalently functionalized SWCNTs, even for chirality-enriched samples, and show the feasibility of applying CE for high-performance separation of nanomaterials based on differences in surface functional density. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Covalently immobilized platelet-derived growth factor-BB promotes angiogenesis in biomimetic poly(ethylene glycol) hydrogels

PubMed Central

Saik, Jennifer E.; Gould, Daniel J.; Watkins, Emily M.; Dickinson, Mary E.; West, Jennifer L.

2011-01-01

The field of tissue engineering is severely limited by a lack of microvascularization in tissue engineered constructs. Biomimetic poly(ethylene glycol) hydrogels containing covalently immobilized platelet-derived growth factor BB (PDGF-BB) were developed to promote angiogenesis. Poly(ethylene glycol) hydrogels resist protein absorption and subsequent non-specific cell adhesion, thus providing a “blank slate”, which can be modified through the incorporation of cell adhesive ligands and growth factors. PDGF-BB is a key angiogenic protein able to support neovessel stabilization by inducing functional anastomoses and recruiting pericytes. Due to the widespread effects of PDGF in the body and a half-life of only 30 min in circulating blood, immobilization of PDGF-BB may be necessary. In this work bioactive, covalently immobilized PDGF-BB was shown to induce tubulogenesis on two-dimensional modified surfaces, migration in three-dimensional (3D) degradable hydrogels and angiogenesis in a mouse cornea micro-pocket angiogenesis assay. Covalently immobilized PDGF-BB was also used in combination with covalently immobilized fibroblast growth factor-2, which led to significantly increased endothelial cell migration in 3D degradable hydrogels compared with the presentation of each factor alone. When a co-culture of endothelial cells and mouse pericyte precursor 10T1/2 cells was seeded onto modified surfaces tubule formation was independent of surface modifications with covalently immobilized growth factors. Furthermore, the combination of soluble PDGF-BB and immobilized PDGF-BB induced a more robust vascular response compared with soluble PDGF-BB alone when implanted into an in vivo mouse cornea micropocket angiogenesis assay. Based on these results, we believe bioactive hydrogels can be tailored to improve the formation of functional microvasculature for tissue engineering. PMID:20801242

Covalent and non-covalent curcumin loading in acid-responsive polymeric micellar nanocarriers

NASA Astrophysics Data System (ADS)

Gao, Min; Chen, Chao; Fan, Aiping; Zhang, Ju; Kong, Deling; Wang, Zheng; Zhao, Yanjun

2015-07-01

Poor aqueous solubility, potential degradation, rapid metabolism and elimination lead to low bioavailability of pleiotropic impotent curcumin. Herein, we report two types of acid-responsive polymeric micelles where curcumin was encapsulated via both covalent and non-covalent modes for enhanced loading capacity and on-demand release. Biodegradable methoxy poly(ethylene glycol)-poly(lactic acid) copolymer (mPEG-PLA) was conjugated with curcumin via a hydrazone linker, generating two conjugates differing in architecture (single-tail versus double-tail) and free curcumin was encapsulated therein. The two micelles exhibited similar hydrodynamic size at 95 ± 3 nm (single-tail) and 96 ± 3 nm (double-tail), but their loading capacities differed significantly at 15.0 ± 0.5% (w/w) (single-tail) and 4.8 ± 0.5% (w/w) (double-tail). Under acidic sink conditions (pH 5.0 and 6.0), curcumin displayed a faster release from the single-tail nanocarrier, which was correlated to a low IC50 of 14.7 ± 1.6 (μg mL-1) compared to the value of double-tail micelle (24.9 ± 1.3 μg mL-1) in HeLa cells. The confocal imaging and flow cytometry analysis demonstrated a superior capability of single-tail micelle for intracellular curcumin delivery, which was a consequence of the higher loading capacity and lower degree of mPEG surface coverage. In conclusion, the dual loading mode is an effective means to increase the drug content in the micellar nanocarriers whose delivery efficiency is highly dependent on its polymer-drug conjugate architecture. This strategy offers an alternative nanoplatform for intracellularly delivering impotent hydrophobic agents (i.e. curcumin) in an efficient stimuli-triggered way, which is valuable for the enhancement of curcumin’s efficacy in managing a diverse range of disorders.

Surface modification of silicon carbide with silane coupling agent and hexadecyl iodiele

NASA Astrophysics Data System (ADS)

Shang, Xujing; Zhu, Yumei; Li, Zhihong

2017-02-01

In this paper, two kinds of silane coupling agents, namely 3-aminopropyl triethoxysilane (KH550) and 3-mercaptopropyl trimethoxysilane (KH590), were adopted as preliminary modifiers to improve the hydrophobic surface properties of silicon carbide (SiC) powder for the first step. The factors that influence the modification effects were investigated by measuring the contact angle. The results showed that KH590 has a better effect than KH550 for the hydrophobic modification of SiC, and the contact angle improved most after SiC powder was reacted with 0.3 g KH590 at 75 °C in aqueous/alcohol solution for 4 h. On account of further enhancement of hydrophobicity, the study was focused on utilizing nucleophilic substitution between KH590 and hexadecyl iodiele to extend the length of alkyl chain. Compared with using KH590 alone, SiC powder modified by KH590 and hexadecyl iodiele showed better water resistance with an increase of contact angle from 106.8° to 127.5°. The Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectra (XPS) as well as X-ray diffraction (XRD) analysis results showed that KH550/KH590 and hexadecyl iodiele can be covalently bonded to the surface of SiC powder without altering its crystal configuration. This methodology may provide a new way of the modification of inorganic materials in further.

Cell behavior on gallium nitride surfaces: peptide affinity attachment versus covalent functionalization.

PubMed

Foster, Corey M; Collazo, Ramon; Sitar, Zlatko; Ivanisevic, Albena

2013-07-02

Gallium nitride is a wide band gap semiconductor that demonstrates a unique set of optical and electrical properties as well as aqueous stability and biocompatibility. This combination of properties makes gallium nitride a strong candidate for use in chemical and biological applications such as sensors and neural interfaces. Molecular modification can be used to enhance the functionality and properties of the gallium nitride surface. Here, gallium nitride surfaces were functionalized with a PC12 cell adhesion promoting peptide using covalent and affinity driven attachment methods. The covalent scheme proceeded by Grignard reaction and olefin metathesis while the affinity driven scheme utilized the recognition peptide isolated through phage display. This study shows that the method of attaching the adhesion peptide influences PC12 cell adhesion and differentiation as measured by cell density and morphological analysis. Covalent attachment promoted monolayer and dispersed cell adhesion while affinity driven attachment promoted multilayer cell agglomeration. Higher cell density was observed on surfaces modified using the recognition peptide. The results suggest that the covalent and affinity driven attachment methods are both suitable for promoting PC12 cell adhesion to the gallium nitride surface, though each method may be preferentially suited for distinct applications.

Crystal structure of the tri-ethyl-ammonium salt of 3-[(4-hy-droxy-3-meth-oxy-phen-yl)(4-hy-droxy-2-oxo-2H-chromen-3-yl)meth-yl]-2-oxo-2H-chromen-4-olate.

PubMed

Ikram, Muhammad; Rehman, Sadia; Khan, Afzal; Schulzke, Carola

2018-03-01

The reaction between 3,3'-[(3-meth-oxy-4-hy-droxy-phen-yl)methanedi-yl]bis-(4-hy-droxy-2 H -chromen-2-one) and tri-ethyl-amine in methanol yielded the title compound tri-ethyl-ammonium 3-[(4-hy-droxy-3-meth-oxy-phen-yl)(4-hy-droxy-2-oxo-2 H -chromen-3-yl)meth-yl]-2-oxo-2 H -chromen-4-olate, C 6 H 16 N + ·C 26 H 17 O 8 - or (NHEt 3 ) + (C 26 H 17 O 8 ) - , which crystallized directly from its methano-lic mother liquor. The non-deprotonated coumarol substituent shares its H atom with the deprotonated coumarolate substituent in a short negative charge-assisted hydrogen bond in which the freely refined H atom is moved from its parent O atom towards the acceptor O atom, elongating the covalent O-H bond to 1.18 (3) Å. The respective H atom can therefore be described as being shared by two alcohol O atoms, culminating in the formation of an eight-membered ring.

H3.Y discriminates between HIRA and DAXX chaperone complexes and reveals unexpected insights into human DAXX-H3.3-H4 binding and deposition requirements

PubMed Central

Zink, Lisa-Maria; Delbarre, Erwan; Eberl, H. Christian; Keilhauer, Eva C.; Bönisch, Clemens; Pünzeler, Sebastian; Bartkuhn, Marek; Collas, Philippe; Mann, Matthias

2017-01-01

Abstract Histone chaperones prevent promiscuous histone interactions before chromatin assembly. They guarantee faithful deposition of canonical histones and functionally specialized histone variants into chromatin in a spatial- and temporally-restricted manner. Here, we identify the binding partners of the primate-specific and H3.3-related histone variant H3.Y using several quantitative mass spectrometry approaches, and biochemical and cell biological assays. We find the HIRA, but not the DAXX/ATRX, complex to recognize H3.Y, explaining its presence in transcriptionally active euchromatic regions. Accordingly, H3.Y nucleosomes are enriched in the transcription-promoting FACT complex and depleted of repressive post-translational histone modifications. H3.Y mutational gain-of-function screens reveal an unexpected combinatorial amino acid sequence requirement for histone H3.3 interaction with DAXX but not HIRA, and for H3.3 recruitment to PML nuclear bodies. We demonstrate the importance and necessity of specific H3.3 core and C-terminal amino acids in discriminating between distinct chaperone complexes. Further, chromatin immunoprecipitation sequencing experiments reveal that in contrast to euchromatic HIRA-dependent deposition sites, human DAXX/ATRX-dependent regions of histone H3 variant incorporation are enriched in heterochromatic H3K9me3 and simple repeat sequences. These data demonstrate that H3.Y's unique amino acids allow a functional distinction between HIRA and DAXX binding and its consequent deposition into open chromatin. PMID:28334823

Microwave plasma induced surface modification of diamond-like carbon films

NASA Astrophysics Data System (ADS)

Rao Polaki, Shyamala; Kumar, Niranjan; Gopala Krishna, Nanda; Madapu, Kishore; Kamruddin, Mohamed; Dash, Sitaram; Tyagi, Ashok Kumar

2017-12-01

Tailoring the surface of diamond-like carbon (DLC) film is technically relevant for altering the physical and chemical properties, desirable for useful applications. A physically smooth and sp3 dominated DLC film with tetrahedral coordination was prepared by plasma-enhanced chemical vapor deposition technique. The surface of the DLC film was exposed to hydrogen, oxygen and nitrogen plasma for physical and chemical modifications. The surface modification was based on the concept of adsorption-desorption of plasma species and surface entities of films. Energetic chemical species of microwave plasma are adsorbed, leading to desorbtion of the surface carbon atoms due to energy and momentum exchange. The interaction of such reactive species with DLC films enhanced the roughness, surface defects and dangling bonds of carbon atoms. Adsorbed hydrogen, oxygen and nitrogen formed a covalent network while saturating the dangling carbon bonds around the tetrahedral sp3 valency. The modified surface chemical affinity depends upon the charge carriers and electron covalency of the adsorbed atoms. The contact angle of chemically reconstructed surface increases when a water droplet interacts either through hydrogen or van dear Waals bonding. These weak interactions influenced the wetting property of the DLC surface to a great extent.

Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9

PubMed Central

LeRoy, Gary; Bua, Dennis J; Garcia, Benjamin A; Gozani, Or; Richard, Stéphane

2010-01-01

Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4me3) while heterochromatin contains the H3K9me3 mark. The H3K9me3 modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4me3. In addition, we show that SETDB1 fails to methylate substrates containing the H3K4me3 mark. Likewise, the functionally related H3K9 KMTs G9A, GLP and SUV39H1 also fail to bind and to methylate H3K4me3 substrates. Accordingly, we provide in vivo evidence that H3K9me2-enriched histones are devoid of H3K4me2/3 and that histones depleted of H3K4me2/3 have elevated H3K9me2/3. The correlation between the loss of interaction of these KMTs with H3K4me3 and concomitant methylation impairment leads to the postulate that at least these four KMTs require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4me3 and H3K9me3. Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners. PMID:21124070

H3K27me3 forms BLOCs over silent genes and intergenic regions and specifies a histone banding pattern on a mouse autosomal chromosome

PubMed Central

Pauler, Florian M.; Sloane, Mathew A.; Huang, Ru; Regha, Kakkad; Koerner, Martha V.; Tamir, Ido; Sommer, Andreas; Aszodi, Andras; Jenuwein, Thomas; Barlow, Denise P.

2009-01-01

In mammals, genome-wide chromatin maps and immunofluorescence studies show that broad domains of repressive histone modifications are present on pericentromeric and telomeric repeats and on the inactive X chromosome. However, only a few autosomal loci such as silent Hox gene clusters have been shown to lie in broad domains of repressive histone modifications. Here we present a ChIP-chip analysis of the repressive H3K27me3 histone modification along chr 17 in mouse embryonic fibroblast cells using an algorithm named broad local enrichments (BLOCs), which allows the identification of broad regions of histone modifications. Our results, confirmed by BLOC analysis of a whole genome ChIP-seq data set, show that the majority of H3K27me3 modifications form BLOCs rather than focal peaks. H3K27me3 BLOCs modify silent genes of all types, plus flanking intergenic regions and their distribution indicates a negative correlation between H3K27me3 and transcription. However, we also found that some nontranscribed gene-poor regions lack H3K27me3. We therefore performed a low-resolution analysis of whole mouse chr 17, which revealed that H3K27me3 is enriched in mega-base-pair-sized domains that are also enriched for genes, short interspersed elements (SINEs) and active histone modifications. These genic H3K27me3 domains alternate with similar-sized gene-poor domains. These are deficient in active histone modifications, as well as H3K27me3, but are enriched for long interspersed elements (LINEs) and long-terminal repeat (LTR) transposons and H3K9me3 and H4K20me3. Thus, an autosome can be seen to contain alternating chromatin bands that predominantly separate genes from one retrotransposon class, which could offer unique domains for the specific regulation of genes or the silencing of autonomous retrotransposons. PMID:19047520

Covalent binding of cyclohexene, 1,3-cyclohexadiene and 1,4-cyclohexadiene on Si(1 1 1)-7 × 7

NASA Astrophysics Data System (ADS)

Tao, Feng; Wang, Zhong Hai; Xu, Guo Qin

2003-05-01

The adsorption reactions and binding configurations of cyclohexene, 1,3-cyclohexadiene and 1,4-cyclohexadiene on Si(1 1 1)-7 × 7 were studied using high-resolution electron energy loss spectroscopy (HREELS), ultraviolet photoelectron spectroscopy (UPS), X-ray photoelectron spectroscopy (XPS) and DFT calculation. The covalent attachments of these unsaturated hydrocarbons to Si(1 1 1)-7 × 7 through the formation of Si-C linkages are clearly demonstrated by the observation of the Si-C stretching mode at 450-500 cm -1 in their HREELS spectra. For chemisorbed cyclohexene, the involvement of π CC in binding is further supported by the absence of CC stretching modes and the disappearance of the π CC photoemission. The chemisorption of both 1,3-cyclohexadiene and 1,4-cyclohexadiene leads to the formation of cyclohexene-like intermediates through di-σ bonding. The existence of one π CC bond in their chemisorbed states is confirmed by the observation of the CC and (sp 2)CH stretching modes and the UPS and XPS results. DFT calculations show that [4 + 2]-like cycloaddition is thermodynamically preferred for 1,3-cyclohexadiene on Si(1 1 1)-7 × 7, but a [2 + 2]-like reaction mechanism is proposed for the covalent attachment of cyclohexene and 1,4-cyclohexadiene.

Evaluation of Heterogeneous Metal-Organic Framework Organocatalysts Prepared by Postsynthetic Modification

PubMed Central

Garibay, Sergio J.; Wang, Zhenqiang; Cohen, Seth M.

2010-01-01

A metal-organic framework (MOF) containing 2-amino-1,4-benzenedicarboxylate (NH2-BDC) as a building block is shown to undergo chemical modification with a set of cyclic anhydrides. The modification of the aluminum-based MOF known as MIL-53(Al)-NH2 (MIL = Matérial Institut Lavoisier) by these reagents is demonstrated by using a variety of methods, including NMR and ESI-MS, and the structural integrity of the modified MOFs has been confirmed by TGA, PXRD, and gas sorption analysis. Reaction with these cyclic anhydrides produces MOFs that display carboxylic acid functional groups within their pores. Furthermore, it is shown that maleic acid functionalized MIL-53(Al)-AMMal can act as a Brønsted acid catalyst and facilitate the methanolysis of several small epoxides. Experiments show that MIL-53(Al)-AMMal acts in a heterogeneous manner and is recyclable with consistent activity over at least three catalytic cycles. The findings presented here demonstrate several important features of covalent postsynthetic modification (PSM) on MOFs, including: 1) facile introduction of catalytic functionality using simple organic reagents (e.g. anhydrides); 2) the ability to utilize and recycle organocatalytic MOFs; 3) control of catalytic activity through choice of functional group. The findings clearly illustrate that covalent postsynthetic modification represents a powerful means to access new MOF compounds that serve as organocatalytic materials. PMID:20698561

Inhibition of murine DNA methyltransferase Dnmt3a by DNA duplexes containing pyrimidine-2(1H)-one.

PubMed

Cherepanova, N A; Zhuze, A L; Gromova, E S

2010-09-01

Here we studied the inhibition of the catalytic domain of Dnmt3a methyltransferase (Dnmt3a-CD) by DNA duplexes containing the mechanism-based inhibitor pyrimidine-2(1H)-one (P) instead of the target cytosine. It has been shown that conjugates of Dnmt3a-CD with P-DNA (DNA containing pyrimidine-2(1H)-one) are not stable to heating at 65°C in 0.1% SDS. The yield of covalent intermediate increases in the presence of the regulatory factor Dnmt3L. The importance of the DNA minor groove for covalent intermediate formation during the methylation reaction catalyzed by Dnmt3a-CD has been revealed. P-DNA was shown to inhibit Dnmt3a-CD; the IC(50) is 830 nM. The competitive mechanism of inhibition of Dnmt3a-CD by P-DNA has been elucidated. It is suggested that therapeutic effect of zebularine could be achieved by inhibition of not only Dnmt1 but also Dnmt3a.

Dimethyl Fumarate Inhibits the Nuclear Factor κB Pathway in Breast Cancer Cells by Covalent Modification of p65 Protein.

PubMed

Kastrati, Irida; Siklos, Marton I; Calderon-Gierszal, Esther L; El-Shennawy, Lamiaa; Georgieva, Gergana; Thayer, Emily N; Thatcher, Gregory R J; Frasor, Jonna

2016-02-12

In breast tumors, activation of the nuclear factor κB (NFκB) pathway promotes survival, migration, invasion, angiogenesis, stem cell-like properties, and resistance to therapy--all phenotypes of aggressive disease where therapy options remain limited. Adding an anti-inflammatory/anti-NFκB agent to breast cancer treatment would be beneficial, but no such drug is approved as either a monotherapy or adjuvant therapy. To address this need, we examined whether dimethyl fumarate (DMF), an anti-inflammatory drug already in clinical use for multiple sclerosis, can inhibit the NFκB pathway. We found that DMF effectively blocks NFκB activity in multiple breast cancer cell lines and abrogates NFκB-dependent mammosphere formation, indicating that DMF has anti-cancer stem cell properties. In addition, DMF inhibits cell proliferation and significantly impairs xenograft tumor growth. Mechanistically, DMF prevents p65 nuclear translocation and attenuates its DNA binding activity but has no effect on upstream proteins in the NFκB pathway. Dimethyl succinate, the inactive analog of DMF that lacks the electrophilic double bond of fumarate, is unable to inhibit NFκB activity. Also, the cell-permeable thiol N-acetyl l-cysteine, reverses DMF inhibition of the NFκB pathway, supporting the notion that the electrophile, DMF, acts via covalent modification. To determine whether DMF interacts directly with p65, we synthesized and used a novel chemical probe of DMF by incorporating an alkyne functionality and found that DMF covalently modifies p65, with cysteine 38 being essential for the activity of DMF. These results establish DMF as an NFκB inhibitor with anti-tumor activity that may add therapeutic value in the treatment of aggressive breast cancers. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Synthesis, characterization and inhibitory activities of (4-N3[3,5-3H]Phe10)PKI(6-22)amide and its precursors: photoaffinity labeling peptides for the active site of cyclic AMP-dependent protein kinase.

PubMed

Katz, B M; Lundquist, L J; Walsh, D A; Glass, D B

1989-06-01

PKI(6-22)amide is a 17 residue peptide corresponding to the active portion of the heat-stable inhibitor of cAMP-dependent protein kinase. The peptide is a potent (Ki = 1.6 nM), competitive inhibitor of the enzyme. The photoreactive peptide analog (4-azidophenylalanine10)PKI(6-22)amide was synthesized in both its non-radiolabeled and tritiated forms by chemical modification of precursor peptides that were prepared by stepwise solid-phase synthesis. (4-Amino[3,5-3H]phenylalanine10)PKI(6-22)amide, the precursor for the radiolabeled arylazide peptide, was obtained by catalytic reduction of the corresponding peptide containing the 3,5-diiodo-4-aminophenylalanine residue at position 10. The purified PKI peptides were analyzed by HPLC, amino acid analysis, and u.v. spectra. In the dark, (4-azidophenylalanine10)PKI(6-22)amide inhibited the catalytic subunit of cAMP-dependent protein kinase with a Ki value of 2.8 nM. The photoreactivity of the arylazide peptide was demonstrated by time-dependent u.v. spectral changes on exposure to light. Photolysis of the catalytic subunit (4-azido[3,5-3H]phenylalanine10)PKI(6-22)amide complex resulted in specific covalent labeling of the enzyme. The data indicate that this peptide is a useful photoaffinity labeling reagent for the active site of the protein kinase.

Genome-wide ChIP-seq mapping and analysis of butyrate-induced H3K9 and H3K27 acetylation and epigenomic landscape alteration in bovine cells

USDA-ARS?s Scientific Manuscript database

Utilizing next-generation sequencing technology, combined with ChIP (Chromatin Immunoprecipitation) technology, we analyzed histone modification (acetylation) induced by butyrate and the large-scale mapping of the epigenomic landscape of normal histone H3 and acetylated histone H3K9 and H3K27. To d...

Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 deoxy-{Delta}{sup 12,14}-prostaglandin J2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mishra, Nandita; Kar, Rekha; Singha, Prajjal K.

2010-04-23

Arachidonic acid derived endogenous electrophile 15d-PGJ2 has gained much attention in recent years due to its potent anti-proliferative and anti-inflammatory actions mediated through thiol modification of cysteine residues in its target proteins. Here, we show that 15d-PGJ2 at 1 {mu}M concentration converts normal mitochondria into large elongated and interconnected mitochondria through direct binding to mitochondrial fission protein Drp1 and partial inhibition of its GTPase activity. Mitochondrial elongation induced by 15d-PGJ2 is accompanied by increased assembly of Drp1 into large oligomeric complexes through plausible intermolecular interactions. The role of decreased GTPase activity of Drp1 in the formation of large oligomeric complexesmore » is evident when Drp1 is incubated with a non-cleavable GTP analog, GTP{gamma}S or by a mutation that inactivated GTPase activity of Drp1 (K38A). The mutation of cysteine residue (Cys644) in the GTPase effector domain, a reported target for modification by reactive electrophiles, to alanine mimicked K38A mutation induced Drp1 oligomerization and mitochondrial elongation, suggesting the importance of cysteine in GED to regulate the GTPase activity and mitochondrial morphology. Interestingly, treatment of K38A and C644A mutants with 15d-PGJ2 resulted in super oligomerization of both mutant Drp1s indicating that 15d-PGJ2 may further stabilize Drp1 oligomers formed by loss of GTPase activity through covalent modification of middle domain cysteine residues. The present study documents for the first time the regulation of a mitochondrial fission activity by a prostaglandin, which will provide clues for understanding the pathological and physiological consequences of accumulation of reactive electrophiles during oxidative stress, inflammation and degeneration.« less

Low Temperature Synthesized H2Ti3O7 Nanotubes with a High CO2 Adsorption Property by Amine Modification.

PubMed

Ota, Misaki; Hirota, Yuichiro; Uchida, Yoshiaki; Sakamoto, Yasuhiro; Nishiyama, Norikazu

2018-06-12

Carbon dioxide (CO 2 ) capture and storage (CCS) technologies have been attracting attention in terms of tackling with global warming. To date, various CO 2 capture technologies including solvents, membranes, cryogenics, and solid adsorbents have been proposed. Currently, a liquid adsorption method for CO 2 using amine solution (monoethanolamine) has been practically used. However, this liquid phase CO 2 adsorption process requires heat regeneration, and it can cause many problems such as corrosion of equipment and degradation of the solution. Meanwhile, solid adsorption methods using porous materials are more advantageous over the liquid method at these points. In this context, we here evaluated if hydrogen titanate (H 2 Ti 3 O 7 ) nanotubes and the surface modification effectively capture CO 2 . For this aim, we first developed a facile synthesis method of H 2 Ti 3 O 7 nanotubes different from any conventional methods. Briefly, they were converted from the precursors-amorphous TiO 2 nanoparticles at room temperature (25 °C). We then determined the outer and the inner diameters of the H 2 Ti 3 O 7 nanotubes as 3.0 and 0.7 nm, respectively. It revealed that both values were much smaller than the reported ones; thus the specific surface area showed the highest value (735 m 2 /g). Next, the outer surface of H 2 Ti 3 O 7 nanotubes was modified using ethylenediamine to examine if CO 2 adsorption capacity increases. The ethylendiamine-modified H 2 Ti 3 O 7 nanotubes showed a higher CO 2 adsorption capacity (50 cm 3 /g at 0 °C, 100 kPa). We finally concluded that the higher CO 2 adsorption capacity could be explained, not only by the high specific surface area of the nanotubes but also by tripartite hydrogen bonding interactions among amines, CO 2 , and OH groups on the surface of H 2 Ti 3 O 7 .

Inhalable Metal-Rich Air Particles and Histone H3K4 Dimethylation and H3K9 Acetylation in a Cross-sectional Study of Steel Workers

PubMed Central

Cantone, Laura; Nordio, Francesco; Hou, Lifang; Apostoli, Pietro; Bonzini, Matteo; Tarantini, Letizia; Angelici, Laura; Bollati, Valentina; Zanobetti, Antonella; Schwartz, Joel; Bertazzi, Pier A.

2011-01-01

Background: Epidemiology investigations have linked exposure to ambient and occupational air particulate matter (PM) with increased risk of lung cancer. PM contains carcinogenic and toxic metals, including arsenic and nickel, which have been shown in in vitro studies to induce histone modifications that activate gene expression by inducing open-chromatin states. Whether inhalation of metal components of PM induces histone modifications in human subjects is undetermined. Objectives: We investigated whether the metal components of PM determined activating histone modifications in 63 steel workers with well-characterized exposure to metal-rich PM. Methods: We determined histone 3 lysine 4 dimethylation (H3K4me2) and histone 3 lysine 9 acetylation (H3K9ac) on histones from blood leukocytes. Exposure to inhalable metal components (aluminum, manganese, nickel, zinc, arsenic, lead, iron) and to total PM was estimated for each study subject. Results: Both H3K4me2 and H3K9ac increased in association with years of employment in the plant (p-trend = 0.04 and 0.006, respectively). H3K4me2 increased in association with air levels of nickel [β = 0.16; 95% confidence interval (CI), 0.03–0.3], arsenic (β = 0.16; 95% CI, 0.02–0.3), and iron (β = 0.14; 95% CI, 0.01–0.26). H3K9ac showed nonsignificant positive associations with air levels of nickel (β = 0.24; 95% CI, –0.02 to 0.51), arsenic (β = 0.21; 95% CI, –0.06 to 0.48), and iron (β = 0.22; 95% CI, –0.03 to 0.47). Cumulative exposures to nickel and arsenic, defined as the product of years of employment by metal air levels, were positively correlated with both H3K4me2 (nickel: β = 0.16; 95% CI, 0.01–0.3; arsenic: β = 0.16; 95% CI, 0.03–0.29) and H3K9ac (nickel: β = 0.27; 95% CI, 0.01–0.54; arsenic: β = 0.28; 95% CI, 0.04–0.51). Conclusions: Our results indicate histone modifications as a novel epigenetic mechanism induced in human subjects by long-term exposure to inhalable nickel and arsenic. PMID

Covalent binding of C3b to tetanus toxin: influence on uptake/internalization of antigen by antigen-specific and non-specific B cells.

PubMed Central

Villiers, M B; Villiers, C L; Jacquier-Sarlin, M R; Gabert, F M; Journet, A M; Colomb, M G

1996-01-01

Antigen opsonization by the C3b fragment of complement is a significant event in the modulation of cell-mediated immune response, but its mechanism is still largely unknown. The structural characteristics of C3b allow it to act as a bifunctional ligand between antigen and cells via their membrane C3b receptors. It was thus of interest to study the influence of the covalent link between C3b and antigen on the fixation and internalization of this antigen by antigen-presenting cells. Tetanus toxin (TT) was used as antigen, either free or covalently linked to C3b (TT-C3b). The antigen-presenting cells were TT-specific (4.2) or non-specific (BL15) Epstein-Barr virus (EBV)-transformed B cells. C3b was found to play an important role in antigen fixation and internalization by both antigen-specific and antigen non-specific cells. Covalent binding of C3b on TT (1) permitted fixation and internalization of this antigen by non-specific cells via their complement receptors; (2) enhanced antigen fixation and resulted in cross-linking between membrane immunoglobulins and complement receptors on antigen-specific cells. The consequences of covalent C3b binding to TT were analysed using antigen-specific and antigen-nonspecific cells. In both cases, a net increase in antigen fixation was observed. At the intracellular level, covalent C3b binding to TT resulted in a large TT incorporation in endosomes of nonspecific cells, similar to that observed in antigen-specific cells. Thus, C3b covalently linked to antigen enlarges the array of B-cell types capable of presenting antigen, including non-specific cells. Images Figure 2 PMID:8958046

Kinetics study of invertase covalently linked to a new functional nanogel.

PubMed

Raj, Lok; Chauhan, Ghanshyam S; Azmi, Wamik; Ahn, J-H; Manuel, James

2011-02-01

Nanogels are promising materials as supports for enzyme immobilization. A new hydrogel comprising of methacrylic acid (MAAc) and N-vinyl pyrrolidone (N-VP) and ethyleneglycol dimethacrylate (EGDMA) was synthesized and converted to nanogel by an emulsification method. Nanogel was further functionalized by Curtius azide reaction for use as support for the covalent immobilization of invertase (Saccharomyces cerevisiae). As-prepared or invertase-immobilized nanogel was characterized by FTIR, XRD, TEM and nitrogen analysis. The characterization of both free and the immobilized-invertase were performed using a spectrophotometric method at 540 nm. The values of V(max), maximum reaction rate, (0.123 unit/mg), k(m), Michaelis constant (7.429 mol/L) and E(a), energy of activation (3.511 kj/mol) for the immobilized-invertase are comparable with those of the free invertase at optimum conditions (time 70 min, pH 6.0 and temperature 45°C). The covalent immobilization enhanced the pH and thermal stability of invertase. The immobilized biocatalyst was efficiently reused up to eight cycles. Copyright © 2010 Elsevier Ltd. All rights reserved.

Histone modifications in the male germ line of Drosophila.

PubMed

Hennig, Wolfgang; Weyrich, Alexandra

2013-02-22

In the male germ line of Drosophila chromatin remains decondensed and highly transcribed during meiotic prophase until it is rapidly compacted. A large proportion of the cell cycle-regulated histone H3.1 is replaced by H3.3, a histone variant encoded outside the histone repeat cluster and not subject to cell cycle controlled expression. We investigated histone modification patterns in testes of D. melanogaster and D. hydei. In somatic cells of the testis envelope and in germ cells these modification patterns differ from those typically seen in eu- and heterochromatin of other somatic cells. During the meiotic prophase some modifications expected in active chromatin are not found or are found at low level. The absence of H4K16ac suggests that dosage compensation does not take place. Certain histone modifications correspond to either the cell cycle-regulated histone H3.1 or to the testis-specific variant H3.3. In spermatogonia we found H3K9 methylation in cytoplasmic histones, most likely corresponding to the H3.3 histone variant. Most histone modifications persist throughout the meiotic divisions. The majority of modifications persist until the early spermatid nuclei, and only a minority further persist until the final chromatin compaction stages before individualization of the spermatozoa. Histone modification patterns in the male germ line differ from expected patterns. They are consistent with an absence of dosage compensation of the X chromosome during the male meiotic prophase. The cell cycle-regulated histone variant H3.1 and H3.3, expressed throughout the cell cycle, also vary in their modification patterns. Postmeiotically, we observed a highly complex pattern of the histone modifications until late spermatid nuclear elongation stages. This may be in part due to postmeiotic transcription and in part to differential histone replacement during chromatin condensation.

Carbon paste electrode with covalently immobilized thionine for electrochemical sensing of hydrogen peroxide

NASA Astrophysics Data System (ADS)

Thenmozhi, K.; Sriman Narayanan, S.

2017-11-01

A water-soluble redox mediator, thionin was covalently immobilized to the functionalized graphite powder and a carbon paste electrode was fabricated from this modified graphite powder. The immobilization procedure proved to be effective in anchoring the thionin mediator in the graphite electrode setup without any leakage problem during the electrochemical studies. The covalent immobilization of the thionin mediator was studied with FT-IR and the electrochemical response of the thionin carbon paste electrode was optimized on varying the supporting electrolyte, pH and scan rate. The modified electrode exhibited well-defined electrocatalytic activity towards the reduction of H2O2 at a lower potential of -0.266 V with good sensitivity. The developed amperometric sensor was efficient towards H2O2 in the linear range from 2.46 × 10-5 M to 4.76 × 10-3 M, with a detection limit of 1.47 × 10-5 M respectively. Important advantages of this sensor are its excellent electrochemical performance, simple fabrication, easy renewability, reproducible analytical results, acceptable accuracy and good operational and long-term stability.

Covalent modification of reduced graphene oxide by means of diazonium chemistry and use as a drug-delivery system.

PubMed

Wei, Guangcheng; Yan, Miaomiao; Dong, Renhao; Wang, Dong; Zhou, Xiangzhu; Chen, Jingfei; Hao, Jingcheng

2012-11-12

Under acidic conditions, reduced graphene oxide (rGO) was functionalized with p-aminobenzoic acid, which formed the diazonium ions through the diazotization with a wet-chemical method. Surfactants or stabilizers were not applied during the diazotization. After the functionalized rGO was treated through mild sonication in aqueous solution, these functionalized rGO sheets were less than two layers, which was determined by atomic force microscopy (AFM) imaging. The water solubility of functionalized rGO after the introduction of polyethyleneimine (PEI) was improved significantly; it was followed by covalent binding of folic acid (FA) molecules to the functionalized rGO to allow us to specifically target CBRH7919 cancer cells by using FA as a receptor. The loading and release behaviors of elsinochrome A (EA) and doxorubicin (DOX) on the functionalized rGO sheets were investigated. The EA loading ratio onto rGO-C(6)H(4)-CO-NH-PEI-NH-CO-FA (abbreviated rGO-PEI-FA, the weight ratio of drug loaded onto rGO-PEI-FA) was approximately 45.56 %, and that of DOX was approximately 28.62 %. It was interesting that the drug release from rGO-PEI-FA was pH- and salt-dependent. The results of cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry (FCM) assays, as well as cell morphology observations) clearly showed that the concentration of rGO-PEI-FA as the drug-delivery composite should be less than 12.5 mg L(-1). The conjugation of DOX and rGO-PEI-FA can enhance the cancer-cell apoptosis effectively and can also push the cancer cells to the vulnerable G2 phase of the cell cycle, which is most sensitive and susceptible to damage by drugs or radiation. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel fluorescence sensor based on covalent immobilization of 3-amino-9-ethylcarbazole by using silver nanoparticles as bridges and carriers.

PubMed

Tan, Shu-Zhen; Hu, Yan-Jun; Gong, Fu-Chun; Cao, Zhong; Xia, Jiao-Yun; Zhang, Ling

2009-03-23

A novel technique of covalent immobilization of indicator dyes in the preparation of fluorescence sensors is developed. Silver nanoparticles are used as bridges and carriers for anchoring indicator dyes. 3-amino-9-ethylcarbazole (AEC) was employed as an example of indicator dyes with terminal amino groups and covalently immobilized onto the outmost surface of a quartz glass slide. First, the glass slide was functionalized by (3-mercaptopropyl) trimethoxysilane (MPS) to form a thiol-terminated self-assembled monolayer, where silver nanoparticles were strongly bound to the surface through covalent bonding. Then, 16-mercaptohexadecanoic acid (MHDA) was self-assembled to bring carboxylic groups onto the surface of silver nanoparticles. A further activation by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) converted the carboxylic groups into succinimide esters. Finally, the active succinimide esters on the surface of silver nanoparticles were reacted with AEC. Thus, AEC was covalently bound to the glass slide and an AEC-immobilized sensor was obtained. The sensor exhibited very satisfactory reproducibility and reversibility, rapid response and no dye-leaching. Rutin can quench the fluorescence intensity of the sensor and be measured by using the sensor. The linear response of the sensor to rutin covers the range from 2.0 x 10(-6) to 1.5 x 10(-4) molL(-1) with a detection limit of 8.0 x 10(-7) molL(-1). The proposed technique may be feasible to the covalent immobilization of other dyes with primary amino groups.

Synthetic Covalently Linked Dimeric Form of H2 Relaxin Retains Native RXFP1 Activity and Has Improved In Vitro Serum Stability

PubMed Central

Nair, Vinojini B.; Bathgate, Ross A. D.; Separovic, Frances; Samuel, Chrishan S.; Hossain, Mohammed Akhter; Wade, John D.

2015-01-01

Human (H2) relaxin is a two-chain peptide member of the insulin superfamily and possesses potent pleiotropic roles including regulation of connective tissue remodeling and systemic and renal vasodilation. These effects are mediated through interaction with its cognate G-protein-coupled receptor, RXFP1. H2 relaxin recently passed Phase III clinical trials for the treatment of congestive heart failure. However, its in vivo half-life is short due to its susceptibility to proteolytic degradation and renal clearance. To increase its residence time, a covalent dimer of H2 relaxin was designed and assembled through solid phase synthesis of the two chains, including a judiciously monoalkyne sited B-chain, followed by their combination through regioselective disulfide bond formation. Use of a bisazido PEG7 linker and “click” chemistry afforded a dimeric H2 relaxin with its active site structurally unhindered. The resulting peptide possessed a similar secondary structure to the native monomeric H2 relaxin and bound to and activated RXFP1 equally well. It had fewer propensities to activate RXFP2, the receptor for the related insulin-like peptide 3. In human serum, the dimer had a modestly increased half-life compared to the monomeric H2 relaxin suggesting that additional oligomerization may be a viable strategy for producing longer acting variants of H2 relaxin. PMID:25685807

Dynamics of H3K27me3 methylation and demethylation in plant development

PubMed Central

Gan, Eng-Seng; Xu, Yifeng; Ito, Toshiro

2015-01-01

Epigenetic regulation controls multiple aspects of the plant development. The N-terminal tail of histone can be differently modified to regulate various chromatin activities. One of them, the trimethylation of histone H3 lysine 27 (H3K27me3) confers a repressive chromatin state with gene silencing. H3K27me3 is dynamically deposited and removed throughout development. While components of the H3K27me3 writer, Polycomb repressive complex 2 (PRC2), have been reported for almost 2 decades, it is only recently that JUMONJI (JMJ) proteins are reported as H3K27me3 demethylases, affirming the dynamic nature of histone modifications. This review highlights recent progress in plant epigenetic research, focusing on the H3K27me3 demethylases. PMID:26313233

Solid-state mAbs and ADCs subjected to heat-stress stability conditions can be covalently modified with buffer and excipient molecules.

PubMed

Valliere-Douglass, John F; Lewis, Patsy; Salas-Solano, Oscar; Jiang, Shan

2015-02-01

We report that a unique type of chemical modification occurs on lyophilized proteins. Freeze-dried mAbs and antibody-drug conjugates (ADCs) can be covalently modified with buffer and excipient molecules on the side chains of Glu, Asp, Thr, and Ser amino acids when subjected to temperature stress. The reaction occurs primarily via condensation of common buffers and excipients such as histidine, tris, trehalose and sucrose, with Glu and Asp carboxylates in the primary sequence of proteins. The reaction was also found to proceed through condensation of carboxylate containing buffers such as citrate, with Thr and Ser hydroxyls in the primary sequence of proteins. Based on the mass of the covalent adducts observed on mAbs and ADCs, it is apparent that the reaction produces water as a product and is thus favored in a low moisture environments such as a lyophilized protein cake. Herein, we present the evidence for the covalent modification of proteins drawn from case studies of in-depth characterization of heat-stressed mAbs and ADCs in the solid state. We also demonstrate how common charge variant assays such as imaged capillary isoelectric focusing and mass spectrometry can be used to monitor this specific class of protein modification. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Acetylated Histone H3K9 is associated with meiotic recombination hotspots, and plays a role in recombination redundantly with other factors including the H3K4 methylase Set1 in fission yeast

PubMed Central

Yamada, Shintaro; Ohta, Kunihiro; Yamada, Takatomi

2013-01-01

Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double-strand break (DSB) formation at hotspots, but the role and precise landscape of involved modifications remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is elevated, and H3K4me3 is not significantly enriched. Mutating H3K9 to non-acetylatable alanine mildly reduced levels of the DSB-inducing protein Rec12 (the fission yeast homologue of Spo11) and DSB at hotspots, indicating that H3K9ac may be involved in DSB formation by enhancing the interaction between Rec12 and hotspots. In addition, we found that the lack of the H3K4 methyltransferase Set1 generally increased Rec12 binding to chromatin but partially reduced DSB formation at some loci, suggesting that Set1 is also involved in DSB formation. These results suggest that meiotic DSB formation is redundantly regulated by multiple chromatin-related factors including H3K9ac and Set1 in fission yeast. PMID:23382177

Multiple-component covalent organic frameworks

PubMed Central

Huang, Ning; Zhai, Lipeng; Coupry, Damien E.; Addicoat, Matthew A.; Okushita, Keiko; Nishimura, Katsuyuki; Heine, Thomas; Jiang, Donglin

2016-01-01

Covalent organic frameworks are a class of crystalline porous polymers that integrate molecular building blocks into periodic structures and are usually synthesized using two-component [1+1] condensation systems comprised of one knot and one linker. Here we report a general strategy based on multiple-component [1+2] and [1+3] condensation systems that enable the use of one knot and two or three linker units for the synthesis of hexagonal and tetragonal multiple-component covalent organic frameworks. Unlike two-component systems, multiple-component covalent organic frameworks feature asymmetric tiling of organic units into anisotropic skeletons and unusually shaped pores. This strategy not only expands the structural complexity of skeletons and pores but also greatly enhances their structural diversity. This synthetic platform is also widely applicable to multiple-component electron donor–acceptor systems, which lead to electronic properties that are not simply linear summations of those of the conventional [1+1] counterparts. PMID:27460607

Multiple-component covalent organic frameworks

NASA Astrophysics Data System (ADS)

Huang, Ning; Zhai, Lipeng; Coupry, Damien E.; Addicoat, Matthew A.; Okushita, Keiko; Nishimura, Katsuyuki; Heine, Thomas; Jiang, Donglin

2016-07-01

Covalent organic frameworks are a class of crystalline porous polymers that integrate molecular building blocks into periodic structures and are usually synthesized using two-component [1+1] condensation systems comprised of one knot and one linker. Here we report a general strategy based on multiple-component [1+2] and [1+3] condensation systems that enable the use of one knot and two or three linker units for the synthesis of hexagonal and tetragonal multiple-component covalent organic frameworks. Unlike two-component systems, multiple-component covalent organic frameworks feature asymmetric tiling of organic units into anisotropic skeletons and unusually shaped pores. This strategy not only expands the structural complexity of skeletons and pores but also greatly enhances their structural diversity. This synthetic platform is also widely applicable to multiple-component electron donor-acceptor systems, which lead to electronic properties that are not simply linear summations of those of the conventional [1+1] counterparts.

Endometriosis Is Characterized by a Distinct Pattern of Histone 3 and Histone 4 Lysine Modifications

PubMed Central

Monteiro, Janice B.; Colón-Díaz, Maricarmen; García, Miosotis; Gutierrez, Sylvia; Colón, Mariano; Seto, Edward; Laboy, Joaquín

2014-01-01

Background: The histone modification patterns in endometriosis have not been fully characterized. This gap in knowledge results in a poor understanding of the epigenetic mechanisms (and potential therapeutic targets) at play. We aimed to (1) assess global acetylation status of histone 3 (H3) and histone 4 (H4), (2) measure levels of H3 and H4 lysine (K) acetylation and methylation, and (3) to identify histone acetylation patterns in promoter regions of candidate genes in tissues from patients and controls. Methods: Global and K-specific acetylation/methylation levels of histones were measured in 24 lesions, 15 endometrium from patients, and 26 endometrium from controls. Chromatin immunoprecipitation (ChIP)–polymerase chain reaction was used to determine the histone acetylation status of the promoter regions of candidate genes in tissues. Results: The lesions were globally hypoacetylated at H3 (but not H4) compared to eutopic endometrium from controls. Lesions had significantly lower levels of H3K9ac and H4K16ac compared to eutopic endometrium from patients and controls. Tissues from patients were hypermethylated at H3K4, H3K9, and H3K27 compared to endometrium from controls. The ChIP analysis showed hypoacetylation of H3/H4 within promoter regions of candidate genes known to be downregulated in endometriosis (e.g., HOXA10, ESR1, CDH1, and p21WAF1/Cip1) in lesions versus control endometrium. The stereoidogenic factor 1 (SF1) promoter region was enriched for acetylated H3 and H4 in lesions versus control tissues, correlating with its reported high expression in lesions. Conclusions: This study describes the histone code of lesions and endometrium from patients with endometriosis and provides support for a possible role of histone modification in modulation of gene expression in endometriosis. PMID:23899551

Covalency in oxidized uranium

NASA Astrophysics Data System (ADS)

Tobin, J. G.; Yu, S.-W.; Qiao, R.; Yang, W. L.; Booth, C. H.; Shuh, D. K.; Duffin, A. M.; Sokaras, D.; Nordlund, D.; Weng, T.-C.

2015-07-01

Using x-ray emission spectroscopy and absorption spectroscopy, it has been possible to directly access the states in the unoccupied conduction bands that are involved with 5 f and 6 d covalency in oxidized uranium. By varying the oxidizing agent, the degree of 5 f covalency can be manipulated and monitored, clearly and irrevocably establishing the importance of 5 f covalency in the electronic structure of the key nuclear fuel, uranium dioxide.

Covalency in oxidized uranium

DOE PAGES

Tobin, J. G.; Yu, S. -W.; Qiao, R.; ...

2015-07-01

Here, using x-ray emission spectroscopy and absorption spectroscopy, it has been possible to directly access the states in the unoccupied conduction bands that are involved with 5f and 6d covalency in oxidized uranium. By varying the oxidizing agent, the degree of 5f covalency can be manipulated and monitored, clearly and irrevocably establishing the importance of 5f covalency in the electronic structure of the key nuclear fuel, uranium dioxide.

Novel nanoparticles based on chitosan-dicarboxylate conjugates via tandem ionotropic/covalent crosslinking with tripolyphosphate and subsequent evaluation as drug delivery vehicles.

PubMed

Dmour, Isra; Taha, Mutasem O

2017-08-30

Chitosan-based nanoparticles prepared by ionotropic gelation are prone to stability issues. The aim of this work is to chemically modify chitosan by grafting to succinate, phthalate, glutarate and phenylsuccinate moieties and to investigate the suitability of the resulting polymers as covalently-crosslinked nanocarriers. Corresponding nanoparticles (NPs) were formulated by ionotropic gelation using tripolyphosphate (TPP) anion then they were covalently crosslinked using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). Infrared and thermal analysis confirmed the formation of phosphoramide bonds within the NPs indicating the involvement of TPP in covalent crosslinking. This is the first time to report phosphormide covalent crosslinking within nanoparticles matrices. The resulting NPs were found to resist drastic pH and calcium ion conditions. Size analysis indicated the NPs to be spherical and less than 500nm in diameter. Loading studies using Safranine O showed enhanced NPs drug loading upon covalent crosslinking compared to ionotropic gelling. Doxorubicin-loaded NPs were of superior cytotoxic properties compared to free doxorubicin. Copyright © 2017 Elsevier B.V. All rights reserved.

Specific histone modification responds to arsenic-induced oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Lu

To explore whether specific histone modifications are associated with arsenic-induced oxidative damage, we recruited 138 arsenic-exposed and arsenicosis subjects from Jiaole Village, Xinren County of Guizhou province, China where the residents were exposed to arsenic from indoor coal burning. 77 villagers from Shang Batian Village that were not exposed to high arsenic coal served as the control group. The concentrations of urine and hair arsenic in the arsenic-exposure group were 2.4-fold and 2.1-fold (all P < 0.001) higher, respectively, than those of the control group. Global histone modifications in human peripheral lymphocytes (PBLCs) were examined by ELISA. The results showedmore » that altered global levels of H3K18ac, H3K9me2, and H3K36me3 correlated with both urinary and hair-arsenic levels of the subjects. Notably, H3K36me3 and H3K18ac modifications were associated with urinary 8-OHdG (H3K36me3: β = 0.16; P = 0.042, H3K18ac: β = − 0.24; P = 0.001). We also found that the modifications of H3K18ac and H3K36me3 were enriched in the promoters of oxidative stress response (OSR) genes in human embryonic kidney (HEK) cells and HaCaT cells, providing evidence that H3K18ac and H3K36me3 modifications mediate transcriptional regulation of OSR genes in response to NaAsO{sub 2} treatment. Particularly, we found that reduced H3K18ac modification correlated with suppressed expression of OSR genes in HEK cells with long term arsenic treatment and in PBLCs of all the subjects. Taken together, we reveal a critical role for specific histone modification in response to arsenic-induced oxidative damage. - Highlights: • H3K18ac, H3K9me2 and H3K36me3 were associated with arsenic exposed levels. • H3K18ac and H3K36me3 were correlated with oxidative damage induced by arsenic. • H3K18ac and H3K36me3 might involve in transcriptional regulation of OSR genes. • Dysregulation of H3K18ac and H3K36me3 might be biomarkers of arsenic toxicity.« less

Role of heme in intracellular trafficking of thyroperoxidase and involvement of H2O2 generated at the apical surface of thyroid cells in autocatalytic covalent heme binding.

PubMed

Fayadat, L; Niccoli-Sire, P; Lanet, J; Franc, J L

1999-04-09

Thyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by approximately 30 and approximately 80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (+120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 8857-8960) that covalent attachment of heme to mammalian peroxidases could be an H2O2-dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H2O2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 microM H2O2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H2O2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H2O2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule.

Bond-bending isomerism of Au 2I 3 -: Competition between covalent bonding and aurophilicity

DOE PAGES

Li, Wan -Lu; Liu, Hong -Tao; Jian, Tian; ...

2015-10-13

We report a joint photoelectron spectroscopy and theoretical investigation of the gaseous Au 2I 3 – cluster, which is found to exhibit two types of isomers due to competition between Au–I covalent bonding and Au–Au aurophilic interactions. The covalent bonding favors a bent IAuIAuI – structure with an obtuse Au–I–Au angle (100.7°), while aurophilic interactions pull the two Au atoms much closer, leading to an acutely bent structure (72.0°) with an Au–Au distance of 3.08 Å. The two isomers are separated by a small barrier and are nearly degenerate with the obtuse isomer being slightly more stable. At low temperature,more » only the obtuse isomer is observed; distinct experimental evidence is observed for the co-existence of a combination of isomers with both acute and obtuse bending angles at room temperature. As a result, the two bond-bending isomers of Au 2I 3 – reveal a unique example of one molecule being able to oscillate between different structures as a result of two competing chemical forces.« less

Bond-bending isomerism of Au 2I 3 -: Competition between covalent bonding and aurophilicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Wan -Lu; Liu, Hong -Tao; Jian, Tian

We report a joint photoelectron spectroscopy and theoretical investigation of the gaseous Au 2I 3 – cluster, which is found to exhibit two types of isomers due to competition between Au–I covalent bonding and Au–Au aurophilic interactions. The covalent bonding favors a bent IAuIAuI – structure with an obtuse Au–I–Au angle (100.7°), while aurophilic interactions pull the two Au atoms much closer, leading to an acutely bent structure (72.0°) with an Au–Au distance of 3.08 Å. The two isomers are separated by a small barrier and are nearly degenerate with the obtuse isomer being slightly more stable. At low temperature,more » only the obtuse isomer is observed; distinct experimental evidence is observed for the co-existence of a combination of isomers with both acute and obtuse bending angles at room temperature. As a result, the two bond-bending isomers of Au 2I 3 – reveal a unique example of one molecule being able to oscillate between different structures as a result of two competing chemical forces.« less

Controlling Interfacial Dynamics: Covalent Bonding versus Physical Adsorption in Polymer Nanocomposites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holt, Adam P.; Bocharova, Vera; Cheng, Shiwang

It is generally believed that the strength of the polymer nanoparticle interaction controls the modification of near-interface segmental mobility in polymer nanocomposites (PNCs). However, little is known about the effect of covalent bonding on the segmental dynamics and glass transition of matrix-free polymer-grafted nanoparticles (PGNs), especially when compared to PNCs. In this article, we directly compare the static and dynamic properties of poly(2-vinylpyridine)/silica-based nanocomposites with polymer chains either physically adsorbed (PNCs) or covalently bonded (PGNs) to identical silica nanoparticles (RNP = 12.5 nm) for three different molecular weight (MW) systems. Interestingly, when the MW of the matrix is as lowmore » as 6 kg/mol (RNP/Rg = 5.4) or as high as 140 kg/mol (RNP/Rg= 1.13), both small-angle X-ray scattering and broadband dielectric spectroscopy show similar static and dynamic properties for PNCs and PGNs. However, for the intermediate MW of 18 kg/mol (RNP/Rg = 3.16), the difference between physical adsorption and covalent bonding can be clearly identified in the static and dynamic properties of the interfacial layer. We ascribe the differences in the interfacial properties of PNCs and PGNs to changes in chain stretching, as quantified by self-consistent field theory calculations. These results demonstrate that the dynamic suppression at the interface is affected by the chain stretching; that is, it depends on the anisotropy of the segmental conformations, more so than the strength of the interaction, which suggests that the interfacial dynamics can be effectively tuned by the degree of stretching a parameter accessible from the MW or grafting density.« less

Controlling Interfacial Dynamics: Covalent Bonding versus Physical Adsorption in Polymer Nanocomposites

DOE PAGES

Holt, Adam P.; Bocharova, Vera; Cheng, Shiwang; ...

2016-06-23

It is generally believed that the strength of the polymer nanoparticle interaction controls the modification of near-interface segmental mobility in polymer nanocomposites (PNCs). However, little is known about the effect of covalent bonding on the segmental dynamics and glass transition of matrix-free polymer-grafted nanoparticles (PGNs), especially when compared to PNCs. In this article, we directly compare the static and dynamic properties of poly(2-vinylpyridine)/silica-based nanocomposites with polymer chains either physically adsorbed (PNCs) or covalently bonded (PGNs) to identical silica nanoparticles (RNP = 12.5 nm) for three different molecular weight (MW) systems. Interestingly, when the MW of the matrix is as lowmore » as 6 kg/mol (RNP/Rg = 5.4) or as high as 140 kg/mol (RNP/Rg= 1.13), both small-angle X-ray scattering and broadband dielectric spectroscopy show similar static and dynamic properties for PNCs and PGNs. However, for the intermediate MW of 18 kg/mol (RNP/Rg = 3.16), the difference between physical adsorption and covalent bonding can be clearly identified in the static and dynamic properties of the interfacial layer. We ascribe the differences in the interfacial properties of PNCs and PGNs to changes in chain stretching, as quantified by self-consistent field theory calculations. These results demonstrate that the dynamic suppression at the interface is affected by the chain stretching; that is, it depends on the anisotropy of the segmental conformations, more so than the strength of the interaction, which suggests that the interfacial dynamics can be effectively tuned by the degree of stretching a parameter accessible from the MW or grafting density.« less

The electronic donation and frequency shifts on the YCCH⋯BH4- boron-bonded complexes (Y = H, CH3, CF3 and CCl3)

NASA Astrophysics Data System (ADS)

Pordeus, Renato Q.; Rego, Danilo G.; Oliveira, Boaz G.

2015-06-01

In this theoretical work, the tetrahydroborate ion (BH4-) was used as proton acceptor in the formation of the YCC-H⋯BH4- complexes (Y = H, CH3, CCl3 and CF3). Using B3LYP/6-311++G(d,p) level of theory, the results of structure corroborate with the analyses of infrared spectra showing that the changes in the bond lengths are in good agreement with the frequency shifts of the HCC-H, H3CCC-H, Cl3CCC-H and F3CCC-H proton donors. Based on the calculations carried out by the Quantum Theory of Atoms in Molecules (QTAIM), the reductions of electronic density corroborate with the red shifts in the frequencies of the C-H bonds. In addition to that, the C-H bonds are polarized because the contributions of s orbital diminish whereas of p increase. In line with this, the variations on the atomic radii computed via QTAIM calculations show that carbon outweigh hydrogen as follows (ΔrC > ΔrH). This scenario is indirectly supported by the Bent's rule of the chemical bonding. Although the interaction energies (corrected with BSSE and ZPE) vary between -19 and -67 kJ mol-1, these complexes interact without covalent character.

Preparation and characterization of malonic acid cross-linked chitosan and collagen 3D scaffolds: an approach on non-covalent interactions.

PubMed

Mitra, Tapas; Sailakshmi, G; Gnanamani, A; Mandal, A B

2012-05-01

The present study emphasizes the influence of non-covalent interactions on the mechanical and thermal properties of the scaffolds of chitosan/collagen origin. Malonic acid (MA), a bifuncitonal diacid was chosen to offer non-covalent cross-linking. Three dimensional scaffolds was prepared using chitosan at 1.0% (w/v) and MA at 0.2% (w/v), similarly collagen 0.5% (w/v) and MA 0.2% (w/v) and characterized. Results on FT-IR, TGA, DSC, SEM and mechanical properties (tensile strength, stiffness, Young's modulus, etc.) assessment demonstrated the existence of non-covalent interaction between MA and chitosan/collagen, which offered flexibility and high strength to the scaffolds suitable for tissue engineering research. Studies using NIH 3T3 fibroblast cells suggested biocompatibility nature of the scaffolds. Docking simulation study further supports the intermolecular hydrogen bonding interactions between MA and chitosan/collagen.

Both H4K20 mono-methylation and H3K56 acetylation mark transcription-dependent histone turnover in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Hanna; Kwon, Chang Seob; Choi, Yoonjung, E-mail: jjungii@kaist.ac.kr

Nucleosome dynamics facilitated by histone turnover is required for transcription as well as DNA replication and repair. Histone turnover is often associated with various histone modifications such as H3K56 acetylation (H3K56Ac), H3K36 methylation (H3K36me), and H4K20 methylation (H4K20me). In order to correlate histone modifications and transcription-dependent histone turnover, we performed genome wide analyses for euchromatic regions in G2/M-arrested fission yeast. The results show that transcription-dependent histone turnover at 5′ promoter and 3′ termination regions is directly correlated with the occurrence of H3K56Ac and H4K20 mono-methylation (H4K20me1) in actively transcribed genes. Furthermore, the increase of H3K56Ac and H4K20me1 and antisense RNAmore » production was observed in the absence of the histone H3K36 methyltransferase Set2 and histone deacetylase complex (HDAC) that are involved in the suppression of histone turnover within the coding regions. These results together indicate that H4K20me1 as well as H3K56Ac are bona fide marks for transcription-dependent histone turnover in fission yeast.« less

Covalent bonding: the fundamental role of the kinetic energy.

PubMed

Bacskay, George B; Nordholm, Sture

2013-08-22

This work addresses the continuing disagreement between two prevalent schools of thought concerning the mechanism of covalent bonding. According to Hellmann, Ruedenberg, and Kutzelnigg, a lowering of the kinetic energy associated with electron delocalization is the key stabilization mechanism. The opposing view of Slater, Feynman, and Bader has maintained that the source of stabilization is electrostatic potential energy lowering due to electron density redistribution to binding regions between nuclei. Despite the large body of accurate quantum chemical work on a range of molecules, the debate concerning the origin of bonding continues unabated, even for H2(+), the simplest of covalently bound molecules. We therefore present here a detailed study of H2(+), including its formation, that uses a sequence of computational methods designed to reveal the relevant contributing mechanisms as well as the spatial density distributions of the kinetic and potential energy contributions. We find that the electrostatic mechanism fails to provide real insight or explanation of bonding, while the kinetic energy mechanism is sound and accurate but complex or even paradoxical to those preferring the apparent simplicity of the electrostatic model. We further argue that the underlying mechanism of bonding is in fact of dynamical character, and analyses that focus on energy do not reveal the origin of covalent bonding in full clarity.

New coordination features; A bridging pyridine and the forced shortest non-covalent distance between two CO 3 2- species

DOE PAGES

Velasco, V.; Aguilà, D.; Barrios, L. A.; ...

2014-09-29

The aerobic reaction of the multidentate ligand 2,6-bis-(3-oxo-3-(2-hydroxyphenyl)-propionyl)-pyridine, H 4L, with Co (II) salts in strong basic conditions produces the clusters [Co 4(L) 2(OH)(py) 7]NO 3 (1) and [Co 8Na 4(L) 4(OH) 2(CO 3) 2(py) 10](BF 4) 2 (2). Analysis of their structure unveils unusual coordination features including a very rare bridging pyridine ligand or two trapped carbonate anions within one coordination cage, forced to stay at an extremely close distance (d O···O = 1.946 Å). This unprecedented non-bonding proximity represents a meeting point between long covalent interactions and “intermolecular” contacts. These original motifs have been analysed here through DFTmore » calculations, which have yielded interaction energies and the reduced repulsion energy experimented by both CO 3 2- anions when located in close proximity inside the coordination cage.« less

Strategies to balance covalent and non-covalent biomolecule attachment within collagen-GAG biomaterials.

PubMed

Pence, Jacquelyn C; Gonnerman, Emily A; Bailey, Ryan C; Harley, Brendan A C

2014-09-01

Strategies to integrate instructive biomolecular signals into a biomaterial are becoming increasingly complex and bioinspired. While a large majority of reports still use repeated treatments with soluble factors, this approach can be prohibitively costly and difficult to translate in vivo for applications where spatial control over signal presentation is necessary. Recent efforts have explored the use of covalent immobilization of biomolecules to the biomaterial, via both bulk (ubiquitous) as well as spatially-selective light-based crosslinking, as a means to both enhance stability and bioactivity. However, little is known about how processing conditions during immobilization impact the degree of unintended non-covalent interactions, or fouling, that takes place between the biomaterial and the biomolecule of interest. Here we demonstrate the impact of processing conditions for bulk carbodiimide (EDC) and photolithography-based benzophenone (BP) crosslinking on specific attachment vs. fouling of a model protein (Concanavalin A, ConA) within collagen-glycosaminoglycan (CG) scaffolds. Collagen source significantly impacts the selectivity of biomolecule immobilization. EDC crosslinking intensity and ligand concentration significantly impacted selective immobilization. For benzophenone photoimmobilization we observed that increased UV exposure time leads to increased ConA immobilization. Immobilization efficiency for both EDC and BP strategies was maximal at physiological pH. Increasing ligand concentration during immobilization process led to enhanced immobilization for EDC chemistry, no impact on BP immobilization, but significant increases in non-specific fouling. Given recent efforts to covalently immobilize biomolecules to a biomaterial surface to enhance bioactivity, improved understanding of the impact of crosslinking conditions on selective attachment versus non-specific fouling will inform the design of instructive biomaterials for applications across tissue

Epigenetic regulation of facultative heterochromatinisation in Planococcus citri via the Me(3)K9H3-HP1-Me(3)K20H4 pathway.

PubMed

Bongiorni, Silvia; Pasqualini, Barbara; Taranta, Monia; Singh, Prim B; Prantera, Giorgio

2007-03-15

Using RNA interference (RNAi) we have conducted a functional analysis of the HP1-like chromobox gene pchet2 during embryogenesis of the mealybug Planococcus citri. Knocking down pchet2 expression results in decondensation of the male-specific chromocenter that normally arises from the developmentally-regulated facultative heterochromatinisation of the paternal chromosome complement. Together with the disappearance of the chromocenter the staining levels of two associated histone modifications, tri-methylated lysine 9 of histone H3 [Me(3)K9H3] and tri-methylated lysine 20 of histone H4 [Me(3)K20H4], are reduced to undetectable levels. Embryos treated with double-stranded RNA (dsRNA) targeting pchet2 also exhibit chromosome abnormalities, such as aberrant chromosome condensation, and also the presence of metaphases that contain 'lagging' chromosomes. We conclude that PCHET2 regulates chromosome behavior during metaphase and is a crucial component of a Me(3)K9H3-HP1-Me(3)K20H4 pathway involved in the facultative heterochromatinisation of the (imprinted) paternal chromosome set.

All-in-One Cellulose Nanocrystals for 3D Printing of Nanocomposite Hydrogels.

PubMed

Wang, Jieping; Chiappone, Annalisa; Roppolo, Ignazio; Shao, Feng; Fantino, Erika; Lorusso, Massimo; Rentsch, Daniel; Dietliker, Kurt; Pirri, Candido Fabrizio; Grützmacher, Hansjörg

2018-02-23

Cellulose nanocrystals (CNCs) with >2000 photoactive groups on each can act as highly efficient initiators for radical polymerizations, cross-linkers, as well as covalently embedded nanofillers for nanocomposite hydrogels. This is achieved by a simple and reliable method for surface modification of CNCs with a photoactive bis(acyl)phosphane oxide derivative. Shape-persistent and free-standing 3D structured objects were printed with a mono-functional methacrylate, showing a superior swelling capacity and improved mechanical properties. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Shifts in podocyte histone H3K27me3 regulate mouse and human glomerular disease

PubMed Central

Majumder, Syamantak; Thieme, Karina; Batchu, Sri N.; Alghamdi, Tamadher A.; Bowskill, Bridgit B.; Kabir, M. Golam; Liu, Youan; Advani, Suzanne L.; White, Kathryn E.; Geldenhuys, Laurette; Tennankore, Karthik K.; Poyah, Penelope; Siddiqi, Ferhan S.

2017-01-01

Histone protein modifications control fate determination during normal development and dedifferentiation during disease. Here, we set out to determine the extent to which dynamic changes to histones affect the differentiated phenotype of ordinarily quiescent adult glomerular podocytes. To do this, we examined the consequences of shifting the balance of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark in podocytes. Adriamycin nephrotoxicity and subtotal nephrectomy (SNx) studies indicated that deletion of the histone methylating enzyme EZH2 from podocytes decreased H3K27me3 levels and sensitized mice to glomerular disease. H3K27me3 was enriched at the promoter region of the Notch ligand Jag1 in podocytes, and derepression of Jag1 by EZH2 inhibition or knockdown facilitated podocyte dedifferentiation. Conversely, inhibition of the Jumonji C domain–containing demethylases Jmjd3 and UTX increased the H3K27me3 content of podocytes and attenuated glomerular disease in adriamycin nephrotoxicity, SNx, and diabetes. Podocytes in glomeruli from humans with focal segmental glomerulosclerosis or diabetic nephropathy exhibited diminished H3K27me3 and heightened UTX content. Analogous to human disease, inhibition of Jmjd3 and UTX abated nephropathy progression in mice with established glomerular injury and reduced H3K27me3 levels. Together, these findings indicate that ostensibly stable chromatin modifications can be dynamically regulated in quiescent cells and that epigenetic reprogramming can improve outcomes in glomerular disease by repressing the reactivation of developmental pathways. PMID:29227285

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.).

PubMed

Ayyappan, Vasudevan; Kalavacharla, Venu; Thimmapuram, Jyothi; Bhide, Ketaki P; Sripathi, Venkateswara R; Smolinski, Tomasz G; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

2015-01-01

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress.

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.)

PubMed Central

Thimmapuram, Jyothi; Bhide, Ketaki P.; Sripathi, Venkateswara R.; Smolinski, Tomasz G.; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

2015-01-01

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress. PMID:26167691

Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry

PubMed Central

Souda, Puneet; Ryan, Christopher M.; Cramer, William A.; Whitelegge, Julian

2011-01-01

Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein’s native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electroncapture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation. PMID:21982782

Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry.

PubMed

Souda, Puneet; Ryan, Christopher M; Cramer, William A; Whitelegge, Julian

2011-12-01

Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein's native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electron-capture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation. Copyright © 2011 Elsevier Inc. All rights reserved.

Reversible covalent binding of neratinib to human serum albumin in vitro.

PubMed

Chandrasekaran, Appavu; Shen, Li; Lockhead, Susan; Oganesian, Aram; Wang, Jianyao; Scatina, JoAnn

2010-12-01

Neratinib (HKI-272), an irreversible inhibitor of Her 2 tyrosine kinase, is currently in development as an alternative for first and second line therapy in metastatic breast cancer patients who overexpress Her 2. Following incubation of [(14)C]neratinib in control human plasma at 37°C for 6 hours, about 60% to 70% of the radioactivity was not extractable, due to covalent binding to albumin. In this study, factors that could potentially affect the covalent binding of neratinib to plasma proteins, specifically to albumin were investigated. When [(14)C]neratinib was incubated at 10 μg/mL in human serum albumin (HSA) or control human plasma, the percent binding increased with time; the highest percentages of binding (46 and 67%, respectively) were observed at 6 hours, the longest duration of incubation examined. Binding increased with increasing temperature; the highest percentages of binding to HSA or human plasma (59 and 78%) were observed at 45°C, the highest temperature tested. The binding also increased with increasing pH of incubation; the highest percentages of binding (56 and 65%) were observed at pH 8.5, the highest pH value tested. The percentages of binding were similar (53% to 57%) when a wide range of concentrations of [(14)C]neratinib (50 ng/mL to 10 μg/mL) were incubated with human plasma at 37°C for 6 hours, indicating that the binding was independent of the substrate concentration, especially in the therapeutic range (50 to 200 ng/mL). When human plasma proteins containing covalently bound [(14)C]neratinb were suspended in a 10 fold volume of phosphate buffer at pH 4.0, 6.0, 7.4, and 8.5, and further incubated at 37°C for ~ 16 hours, about 45%, 44%, 32%, and 12% of the total radioactivity, respectively, was released as unchanged [(14)C]neratinib, indicating that the binding is reversible in nature, with more released at pH 7.4 and below. In conclusion, the covalent binding of neratinib to serum albumin is pH, time and temperature dependent, but

Comparative Analyses of H3K4 and H3K27 Trimethylations Between the Mouse Cerebrum and Testis

PubMed Central

Cui, Peng; Liu, Wanfei; Zhao, Yuhui; Lin, Qiang; Zhang, Daoyong; Ding, Feng; Xin, Chengqi; Zhang, Zhang; Song, Shuhui; Sun, Fanglin; Yu, Jun; Hu, Songnian

2012-01-01

The global features of H3K4 and H3K27 trimethylations (H3K4me3 and H3K27me3) have been well studied in recent years, but most of these studies were performed in mammalian cell lines. In this work, we generated the genome-wide maps of H3K4me3 and H3K27me3 of mouse cerebrum and testis using ChIP-seq and their high-coverage transcriptomes using ribominus RNA-seq with SOLiD technology. We examined the global patterns of H3K4me3 and H3K27me3 in both tissues and found that modifications are closely-associated with tissue-specific expression, function and development. Moreover, we revealed that H3K4me3 and H3K27me3 rarely occur in silent genes, which contradicts the findings in previous studies. Finally, we observed that bivalent domains, with both H3K4me3 and H3K27me3, existed ubiquitously in both tissues and demonstrated an invariable preference for the regulation of developmentally-related genes. However, the bivalent domains tend towards a “winner-takes-all” approach to regulate the expression of associated genes. We also verified the above results in mouse ES cells. As expected, the results in ES cells are consistent with those in cerebrum and testis. In conclusion, we present two very important findings. One is that H3K4me3 and H3K27me3 rarely occur in silent genes. The other is that bivalent domains may adopt a “winner-takes-all” principle to regulate gene expression. PMID:22768982

Oriented and covalent immobilization of target molecules to solid supports: synthesis and application of a light-activatable and thiol-reactive cross-linking reagent.

PubMed

Collioud, A; Clémence, J F; Sänger, M; Sigrist, H

1993-01-01

Light-dependent oriented and covalent immobilization of target molecules has been achieved by combining two modification procedures: light-dependent coupling of target molecules to inert surfaces and thiol-selective reactions occurring at macromolecule or substrate surfaces. For immobilization purposes the heterobifunctional reagent N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide was synthesized and chemically characterized. The photosensitivity of the carbene-generating reagent and its reactivity toward thiols were ascertained. Light-induced cross-linking properties of the reagent were documented (i) by reacting first the maleimide function with a thiolated surface, followed by carbene insertion into applied target molecules, (ii) by photochemical coupling of the reagent to an inert support followed by thermochemical reactions with thiol functions, and (iii) by thermochemical modification of target molecules prior to carbene-mediated insertion into surface materials. Procedures mentioned led to light-dependent covalent immobilization of target molecules including amino acids, a synthetic peptide, and antibody-derived F(ab') fragments. Topically selective, light-dependent immobilization was attained with the bifunctional reagent by irradiation of coated surfaces through patterned masks. Glass and polystyrene served as substrates. Molecular orientation is asserted by inherently available or selectively introduced terminal thiol functions in F(ab') fragments and synthetic polypeptides, respectively.

Covalent Docking of Large Libraries for the Discovery of Chemical Probes

PubMed Central

London, Nir; Miller, Rand M.; Krishnan, Shyam; Uchida, Kenji; Irwin, John J.; Eidam, Oliv; Gibold, Lucie; Cimermančič, Peter; Bonnet, Richard; Shoichet, Brian K.; Taunton, Jack

2014-01-01

Chemical probes that form a covalent bond with a protein target often show enhanced selectivity, potency, and utility for biological studies. Despite these advantages, protein-reactive compounds are usually avoided in high-throughput screening campaigns. Here we describe a general method (DOCKovalent) for screening large virtual libraries of electrophilic small molecules. We apply this method prospectively to discover reversible covalent fragments that target distinct protein nucleophiles, including the catalytic serine of AmpC β-lactamase and noncatalytic cysteines in RSK2, MSK1, and JAK3 kinases. We identify submicromolar to low-nanomolar hits with high ligand efficiency, cellular activity and selectivity, including the first reported reversible covalent inhibitors of JAK3. Crystal structures of inhibitor complexes with AmpC and RSK2 confirm the docking predictions and guide further optimization. As covalent virtual screening may have broad utility for the rapid discovery of chemical probes, we have made the method freely available through an automated web server (http://covalent.docking.org). PMID:25344815

Covalent docking of large libraries for the discovery of chemical probes.

PubMed

London, Nir; Miller, Rand M; Krishnan, Shyam; Uchida, Kenji; Irwin, John J; Eidam, Oliv; Gibold, Lucie; Cimermančič, Peter; Bonnet, Richard; Shoichet, Brian K; Taunton, Jack

2014-12-01

Chemical probes that form a covalent bond with a protein target often show enhanced selectivity, potency and utility for biological studies. Despite these advantages, protein-reactive compounds are usually avoided in high-throughput screening campaigns. Here we describe a general method (DOCKovalent) for screening large virtual libraries of electrophilic small molecules. We apply this method prospectively to discover reversible covalent fragments that target distinct protein nucleophiles, including the catalytic serine of AmpC β-lactamase and noncatalytic cysteines in RSK2, MSK1 and JAK3 kinases. We identify submicromolar to low-nanomolar hits with high ligand efficiency, cellular activity and selectivity, including what are to our knowledge the first reported reversible covalent inhibitors of JAK3. Crystal structures of inhibitor complexes with AmpC and RSK2 confirm the docking predictions and guide further optimization. As covalent virtual screening may have broad utility for the rapid discovery of chemical probes, we have made the method freely available through an automated web server (http://covalent.docking.org/).

Binding matter with antimatter: the covalent positron bond.

PubMed

Charry, Jorge Alfonso; Varella, Marcio T Do N; Reyes, Andrés

2018-05-16

We report sufficient theoretical evidence of the energy stability of the e⁺H₂²⁻ molecule, formed by two H⁻ anions and one positron. Analysis of the electronic and positronic densities of the latter compound undoubtedly points out the formation of a positronic covalent bond between the otherwise repelling hydride anions. The lower limit for the bonding energy of the e⁺H₂²⁻ molecule is 74 kJ/mol (0.77 eV), accounting for the zero-point vibrational correction. The formation of a non electronic covalent bond is fundamentally distinct from positron attachment to stable molecules, as the latter process is characterized by a positron affinity, analogous to the electron affinity. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nevirapine bioactivation and covalent binding in the skin.

PubMed

Sharma, Amy M; Klarskov, Klaus; Uetrecht, Jack

2013-03-18

Nevirapine (NVP) treatment is associated with serious skin rashes that appear to be immune-mediated. We previously developed a rat model of this skin rash that is immune-mediated and is very similar to the rash in humans. Treatment of rats with the major NVP metabolite, 12-OH-NVP, also caused the rash. Most idiosyncratic drug reactions are caused by reactive metabolites; 12-OH-NVP forms a benzylic sulfate, which was detected in the blood of animals treated with NVP or 12-OH-NVP. This sulfate is presumably formed in the liver; however, the skin also has significant sulfotransferase activity. In this study, we used a serum against NVP to detect covalent binding in the skin of rats. There was a large artifact band in immunoblots of whole skin homogenates that interfered with detection of covalent binding; however, when the skin was separated into dermal and epidermal fractions, covalent binding was clearly present in the epidermis, which is also the location of sulfotransferases. In contrast to rats, treatment of mice with NVP did not result in covalent binding in the skin or skin rash. Although the reaction of 12-OH-NVP sulfate with nucleophiles such as glutathione is slow, incubation of this sulfate with homogenized human and rat skin led to extensive covalent binding. Incubations of 12-OH-NVP with the soluble fraction from a 9,000g centrifugation (S9) of rat or human skin homogenate in the presence of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) produced extensive covalent binding, but no covalent binding was detected with mouse skin S9, which suggests that the reason mice do not develop a rash is that they lack the required sulfotransferase. This is the first study to report covalent binding of NVP to rat and human skin. These data provide strong evidence that covalent binding of NVP in the skin is due to 12-OH-NVP sulfate, which is likely responsible for NVP-induced skin rash. Sulfation may represent a bioactivation pathway for other drugs that cause a skin rash.

Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity.

PubMed

Lu, Jun; Zhang, Xiaoli; Shen, Tingting; Ma, Chao; Wu, Jun; Kong, Hualei; Tian, Jing; Shao, Zhifeng; Zhao, Xiaodong; Xu, Ling

2016-01-01

Traditional Chinese medicine Jinfukang (JFK) has been clinically used for treating lung cancer. To examine whether epigenetic modifications are involved in its anticancer activity, we performed a global profiling analysis of H3K4Me3, an epigenomic marker associated with active gene expression, in JFK-treated lung cancer cells. We identified 11,670 genes with significantly altered status of H3K4Me3 modification following JFK treatment (P < 0.05). Gene Ontology analysis indicates that these genes are involved in tumor-related pathways, including pathway in cancer, basal cell carcinoma, apoptosis, induction of programmed cell death, regulation of transcription (DNA-templated), intracellular signal transduction, and regulation of peptidase activity. In particular, we found that the levels of H3K4Me3 at the promoters of SUSD2, CCND2, BCL2A1, and TMEM158 are significantly altered in A549, NCI-H1975, NCI-H1650, and NCI-H2228 cells, when treated with JFK. Collectively, these findings provide the first evidence that the anticancer activity of JFK involves modulation of histone modification at many cancer-related gene loci.

Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity

PubMed Central

Lu, Jun; Zhang, Xiaoli; Shen, Tingting; Ma, Chao; Wu, Jun; Kong, Hualei; Tian, Jing; Shao, Zhifeng; Zhao, Xiaodong; Xu, Ling

2016-01-01

Traditional Chinese medicine Jinfukang (JFK) has been clinically used for treating lung cancer. To examine whether epigenetic modifications are involved in its anticancer activity, we performed a global profiling analysis of H3K4Me3, an epigenomic marker associated with active gene expression, in JFK-treated lung cancer cells. We identified 11,670 genes with significantly altered status of H3K4Me3 modification following JFK treatment (P < 0.05). Gene Ontology analysis indicates that these genes are involved in tumor-related pathways, including pathway in cancer, basal cell carcinoma, apoptosis, induction of programmed cell death, regulation of transcription (DNA-templated), intracellular signal transduction, and regulation of peptidase activity. In particular, we found that the levels of H3K4Me3 at the promoters of SUSD2, CCND2, BCL2A1, and TMEM158 are significantly altered in A549, NCI-H1975, NCI-H1650, and NCI-H2228 cells, when treated with JFK. Collectively, these findings provide the first evidence that the anticancer activity of JFK involves modulation of histone modification at many cancer-related gene loci. PMID:27087825

The increase of apatite layer formation by the poly(3-hydroxybutyrate) surface modification of hydroxyapatite and β-tricalcium phosphate.

PubMed

Szubert, M; Adamska, K; Szybowicz, M; Jesionowski, T; Buchwald, T; Voelkel, A

2014-01-01

The aim of this study was the surface modification of hydroxyapatite and β-tricalcium phosphate by poly(3-hydroxybutyrate) grafting and characterization of modificates. The bioactivity examination was carried out by the determination to grow an apatite layer on modified materials during incubation in simulated body fluid at 37°C. The additional issue taken up in this paper was to investigate the influence of fluid replacement. The process of the surface modification of biomaterials was evaluated by means of infrared and Raman spectroscopy. Formation of the apatite layer was assessed by means of scanning electron microscopy and confirmed by energy dispersive, Raman and Fourier transformed infrared spectroscopy. During exposure in simulated body fluid, the variation of the zeta potential, pH measurement and relative weight was monitored. Examination of scanning electron microscopy micrographs suggests that modification of hydroxyapatite and β-tricalcium phosphate by poly(3-hydroxybutyrate) significantly increases apatite layer formation. Raman spectroscopy evaluation revealed that the formation of the apatite layer was more significant in the case of hydroxyapatite modificate, when compared to the β-tricalcium phosphate modificate. Both modificates were characterized by stable pH, close to the natural pH of human body fluids. Furthermore, we have shown that a weekly changed, simulated body fluid solution increases apatite layer formation. © 2013.

Covalent adaptable networks: smart, reconfigurable and responsive network systems.

PubMed

Kloxin, Christopher J; Bowman, Christopher N

2013-09-07

Covalently crosslinked materials, classically referred to as thermosets, represent a broad class of elastic materials that readily retain their shape and molecular architecture through covalent bonds that are ubiquitous throughout the network structure. These materials, in particular in their swollen gel state, have been widely used as stimuli responsive materials with their ability to change volume in response to changes in temperature, pH, or other solvent conditions and have also been used in shape memory applications. However, the existence of a permanent, unalterable shape and structure dictated by the covalently crosslinked structure has dramatically limited their abilities in this and many other areas. These materials are not generally reconfigurable, recyclable, reprocessable, and have limited ability to alter permanently their stress state, topography, topology, or structure. Recently, a new paradigm has been explored in crosslinked polymers - that of covalent adaptable networks (CANs) in which covalently crosslinked networks are formed such that triggerable, reversible chemical structures persist throughout the network. These reversible covalent bonds can be triggered through molecular triggers, light or other incident radiation, or temperature changes. Upon application of this stimulus, rather than causing a temporary shape change, the CAN structure responds by permanently adjusting its structure through either reversible addition/condensation or through reversible bond exchange mechanisms, either of which allow the material to essentially reequilibrate to its new state and condition. Here, we provide a tutorial review on these materials and their responsiveness to applied stimuli. In particular, we review the broad classification of these materials, the nature of the chemical bonds that enable the adaptable structure, how the properties of these materials depend on the reversible structure, and how the application of a stimulus causes these materials to

Chemical stability of insulin. 3. Influence of excipients, formulation, and pH.

PubMed

Brange, J; Langkjaer, L

1992-01-01

The influence of auxiliary substances and pH on the chemical transformations of insulin in pharmaceutical formulation, including various hydrolytic and intermolecular cross-linking reactions, was studied. Bacteriostatic agents had a profound stabilizing effect--phenol > m-cresol > methylparaben--on deamidation as well as on insulin intermolecular cross-linking reactions. Of the isotonicity substances, NaCl generally had a stabilizing effect whereas glycerol and glucose led to increased chemical deterioration. Phenol and sodium chloride exerted their stabilizing effect through independent mechanisms. Zinc ions, in concentrations that promote association of insulin into hexamers, increase the stability, whereas higher zinc content had no further influence. Protamine gave rise to additional formation of covalent protamine-insulin products which increased with increasing protamine concentration. The impact of excipients on the chemical processes seems to be dictated mainly via an influence on the three-dimensional insulin structure. The effect of the physical state of the insulin on the chemical stability was also complex, suggesting an intricate dependence of intermolecular proximity of involved functional groups. At pH values below five and above eight, insulin degrades relatively fast. At acid pH, deamidation at residue A21 and covalent insulin dimerization dominates, whereas disulfide reactions leading to covalent polymerization and formation of A- and B-chains prevailed in alkaline medium. Structure-reactivity relationship is proposed to be a main determinant for the chemical transformation of insulin.

Simultaneous covalent and noncovalent hybrid polymerizations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Z.; Tantakitti, F.; Yu, T.

Covalent and supramolecular polymers are two distinct forms of soft matter, composed of long chains of covalently and noncovalently linked structural units, respectively. We report a hybrid system formed by simultaneous covalent and supramolecular polymerizations of monomers. The process yields cylindrical fibers of uniform diameter that contain covalent and supramolecular compartments, a morphology not observed when the two polymers are formed independently. The covalent polymer has a rigid aromatic imine backbone with helicoidal conformation, and its alkylated peptide side chains are structurally identical to the monomer molecules of supramolecular polymers. In the hybrid system, covalent chains grow to higher averagemore » molar mass relative to chains formed via the same polymerization in the absence of a supramolecular compartment. The supramolecular compartments can be reversibly removed and re-formed to reconstitute the hybrid structure, suggesting soft materials with novel delivery or repair functions.« less

Photodissociative Cross-Linking of Non-covalent Peptide-Peptide Ion Complexes in the Gas Phase

NASA Astrophysics Data System (ADS)

Nguyen, Huong T. H.; Andrikopoulos, Prokopis C.; Rulíšek, Lubomír; Shaffer, Christopher J.; Tureček, František

2018-05-01

We report a gas-phase UV photodissociation study investigating non-covalent interactions between neutral hydrophobic pentapeptides and peptide ions incorporating a diazirine-tagged photoleucine residue. Phenylalanine (Phe) and proline (Pro) were chosen as the conformation-affecting residues that were incorporated into a small library of neutral pentapeptides. Gas-phase ion-molecule complexes of these peptides with photo-labeled pentapeptides were subjected to photodissociation. Selective photocleavage of the diazirine ring at 355 nm formed short-lived carbene intermediates that underwent cross-linking by insertion into H-X bonds of the target peptide. The cross-link positions were established from collision-induced dissociation tandem mass spectra (CID-MS3) providing sequence information on the covalent adducts. Effects of the amino acid residue (Pro or Phe) and its position in the target peptide sequence were evaluated. For proline-containing peptides, interactions resulting in covalent cross-links in these complexes became more prominent as proline was moved towards the C-terminus of the target peptide sequence. The photocross-linking yields of phenylalanine-containing peptides depended on the position of both phenylalanine and photoleucine. Density functional theory calculations were used to assign structures of low-energy conformers of the (GLPMG + GLL*LK + H)+ complex. Born-Oppenheimer molecular dynamics trajectory calculations were used to capture the thermal motion in the complexes within 100 ps and determine close contacts between the incipient carbene and the H-X bonds in the target peptide. This provided atomic-level resolution of potential cross-links that aided spectra interpretation and was in agreement with experimental data. [Figure not available: see fulltext.

Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity

PubMed Central

Hosogane, Masaki; Funayama, Ryo; Nishida, Yuichiro; Nagashima, Takeshi; Nakayama, Keiko

2013-01-01

Oncogenic signaling pathways regulate gene expression in part through epigenetic modification of chromatin including DNA methylation and histone modification. Trimethylation of histone H3 at lysine-27 (H3K27), which correlates with transcriptional repression, is regulated by an oncogenic form of the small GTPase Ras. Although accumulation of trimethylated H3K27 (H3K27me3) has been implicated in transcriptional regulation, it remains unclear whether Ras-induced changes in H3K27me3 are a trigger for or a consequence of changes in transcriptional activity. We have now examined the relation between H3K27 trimethylation and transcriptional regulation by Ras. Genome-wide analysis of H3K27me3 distribution and transcription at various times after expression of oncogenic Ras in mouse NIH 3T3 cells identified 115 genes for which H3K27me3 level at the gene body and transcription were both regulated by Ras. Similarly, 196 genes showed Ras-induced changes in transcription and H3K27me3 level in the region around the transcription start site. The Ras-induced changes in transcription occurred before those in H3K27me3 at the genome-wide level, a finding that was validated by analysis of individual genes. Depletion of H3K27me3 either before or after activation of Ras signaling did not affect the transcriptional regulation of these genes. Furthermore, given that H3K27me3 enrichment was dependent on Ras signaling, neither it nor transcriptional repression was maintained after inactivation of such signaling. Unexpectedly, we detected unannotated transcripts derived from intergenic regions at which the H3K27me3 level is regulated by Ras, with the changes in transcript abundance again preceding those in H3K27me3. Our results thus indicate that changes in H3K27me3 level in the gene body or in the region around the transcription start site are not a trigger for, but rather a consequence of, changes in transcriptional activity. PMID:24009517

Surface modification of single crystal LiTaO3 by H and He implantation

NASA Astrophysics Data System (ADS)

Ma, Changdong; Lu, Fei; Jin, Lei; Xu, Bo; Fan, Ranran

2017-02-01

Defects production and evolution in H and He ions co-implanted LiTaO3 under different implantation order (H + He and He + H) are investigated. Rutherford backscattering spectrometry (RBS), infrared (IR) spectroscopy and transmission electron microscopy (TEM) are used to study the lattice damage, composition and structure change in the buried damage region. Obvious differences of ions aggregation mechanism are found in H and He implanted LiTaO3. Blistering or splitting of LiTaO3 is more easily achieved in the case where He is implanted first compared to the reverses case. Significant damage enhancement and micro-fractures are observed in samples with He preimplant. The dispersed damage in H-first sample is due to the destruction by He post-bombardment of H-clusters. This order effect indicates the strong aggregation and trapping ability of He ions and He bubbles. The effect of coimplantation parameters on the cleaving of LiTaO3 is discussed.

Covalent intermolecular interaction of the nitric oxide dimer (NO)2

NASA Astrophysics Data System (ADS)

Zhang, Hui; Zheng, Gui-Li; Lv, Gang; Geng, Yi-Zhao; Ji, Qing

2015-09-01

Covalent bonds arise from the overlap of the electronic clouds in the internucleus region, which is a pure quantum effect and cannot be obtained in any classical way. If the intermolecular interaction is of covalent character, the result from direct applications of classical simulation methods to the molecular system would be questionable. Here, we analyze the special intermolecular interaction between two NO molecules based on quantum chemical calculation. This weak intermolecular interaction, which is of covalent character, is responsible for the formation of the NO dimer, (NO)2, in its most stable conformation, a cis conformation. The natural bond orbital (NBO) analysis gives an intuitive illustration of the formation of the dimer bonding and antibonding orbitals concomitant with the breaking of the π bonds with bond order 0.5 of the monomers. The dimer bonding is counteracted by partially filling the antibonding dimer orbital and the repulsion between those fully or nearly fully occupied nonbonding dimer orbitals that make the dimer binding rather weak. The direct molecular mechanics (MM) calculation with the UFF force fields predicts a trans conformation as the most stable state, which contradicts the result of quantum mechanics (QM). The lesson from the investigation of this special system is that for the case where intermolecular interaction is of covalent character, a specific modification of the force fields of the molecular simulation method is necessary. Project supported by the National Natural Science Foundation of China (Grant Nos. 90403007 and 10975044), the Key Subject Construction Project of Hebei Provincial Universities, China, the Research Project of Hebei Education Department, China (Grant Nos. Z2012067 and Z2011133), the National Natural Science Foundation of China (Grant No. 11147103), and the Open Project Program of State Key Laboratory of Theoretical Physics, Institute of Theoretical Physics, Chinese Academy of Sciences, China (Grant No. Y5

Enhanced stability of catalase covalently immobilized on functionalized titania submicrospheres.

PubMed

Wu, Hong; Liang, Yanpeng; Shi, Jiafu; Wang, Xiaoli; Yang, Dong; Jiang, Zhongyi

2013-04-01

In this study, a novel approach combing the chelation and covalent binding was explored for facile and efficient enzyme immobilization. The unique capability of titania to chelate with catecholic derivatives at ambient conditions was utilized for titania surface functionalization. The functionalized titania was then used for enzyme immobilization. Titania submicrospheres (500-600 nm) were synthesized by a modified sol-gel method and functionalized with carboxylic acid groups through a facile chelation method by using 3-(3,4-dihydroxyphenyl) propionic acid as the chelating agent. Then, catalase (CAT) was covalently immobilized on these functionalized titania submicrospheres through 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. The immobilized CAT retained 65% of its free form activity with a loading capacity of 100-150 mg/g titania. The pH stability, thermostability, recycling stability and storage stability of the immobilized CAT were evaluated. A remarkable enhancement in enzyme stability was achieved. The immobilized CAT retained 90% and 76% of its initial activity after 10 and 16 successive cycles of decomposition of hydrogen peroxide, respectively. Both the Km and the Vmax values of the immobilized CAT (27.4 mM, 13.36 mM/min) were close to those of the free CAT (25.7 mM, 13.46 mM/min). Copyright © 2012 Elsevier B.V. All rights reserved.

Intricate Effects of α-Amino and Lysine Modifications on Arginine Methylation of the N-Terminal Tail of Histone H4.

PubMed

Fulton, Melody D; Zhang, Jing; He, Maomao; Ho, Meng-Chiao; Zheng, Y George

2017-07-18

Chemical modifications of the DNA and nucleosomal histones tightly control the gene transcription program in eukaryotic cells. The "histone code" hypothesis proposes that the frequency, combination, and location of post-translational modifications (PTMs) of the core histones compose a complex network of epigenetic regulation. Currently, there are at least 23 different types and >450 histone PTMs that have been discovered, and the PTMs of lysine and arginine residues account for a crucial part of the histone code. Although significant progress has been achieved in recent years, the molecular basis for the histone code is far from being fully understood. In this study, we investigated how naturally occurring N-terminal acetylation and PTMs of histone H4 lysine-5 (H4K5) affect arginine-3 methylation catalyzed by both type I and type II PRMTs at the biochemical level. Our studies found that acylations of H4K5 resulted in decreased levels of arginine methylation by PRMT1, PRMT3, and PRMT8. In contrast, PRMT5 exhibits an increased rate of arginine methylation upon H4K5 acetylation, propionylation, and crotonylation, but not upon H4K5 methylation, butyrylation, or 2-hydroxyisobutyrylation. Methylation of H4K5 did not affect arginine methylation by PRMT1 or PRMT5. There was a small increase in the rate of arginine methylation by PRMT8. Strikingly, a marked increase in the rate of arginine methylation was observed for PRMT3. Finally, N-terminal acetylation reduced the rate of arginine methylation by PRMT3 but had little influence on PRMT1, -5, and -8 activity. These results together highlight the underlying mechanistic differences in substrate recognition among different PRMTs and pave the way for the elucidation of the complex interplay of histone modifications.

Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin

PubMed Central

Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun

2016-01-01

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2. Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. PMID:27534820

Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin.

PubMed

Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun; Chung, Kwang Chul

2016-08-01

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2 Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. © 2016 The Authors.

The effect of hydrochloric acid addition to increase carbon nanotubes dispersibility as drug delivery system by covalent functionalization

NASA Astrophysics Data System (ADS)

Wulan, P. P. D. K.; Ulwani, S. H.; Wulandari, H.; Purwanto, W. W.; Mulia, K.

2018-03-01

This study is to obtain the effect of adding hydrochloric acid (HCl) to the mixture of sulfuric acid (H2SO4) and HNO3 in CNT covalent functionalization. HCl expected to increase the dispersibility of functionalized CNT by improving the dispersion time period done with H2SO4 or HNO3. Functionalization used mixture of H2SO4 and HNO3 with volume ratio of 3:1. Covalent functionalization used 0.5 grams of MWCNT ultra sonicated in 50mL HNO3 with and mixture of H2SO4 and HNO3. Additions of 200 mL HCl used variation of molarity from 1M, 2M, 3M, 4M, 5M to 6M. CNT were oxidized to form carboxylic and hydroxyl bonds on the surface that increase dispersibility of CNT. FTIR spectrums showed the existences of carboxyl and hydroxyl group on spectra of 2600-3700 cm-1 and 900-1400 cm-1. Dispersion tests, which showed functionalized CNT (f-CNT) dispersion capabilities, were done by dissolving f-CNT in water. The study resulted that 6M f-CNT (NSC6) gave the best dispersion with zeta potential value of -37.1mV. NSC6 gave the longest dispersion time which was 20 days until f-CNT settle again. SEM-EDS micrographs showed the surface structure of 6M f-CNT without significant damage and no longer contain impurities of Fe, Ni, and Cl.

Covalent chromatography. Preparation of fully active papain from dried papaya latex

PubMed Central

Brocklehurst, Keith; Carlsson, Jan; Kierstan, Marek P. J.; Crook, Eric M.

1973-01-01

1. A Sepharose–(glutathione–2-pyridyl disulphide) conjugate has been prepared. 2. Its use in a new type of chromatography, covalent chromatography by thiol–disulphide interchange, is described. 3. With this technique, papain containing 1 intact catalytic site [thiol with high reactivity towards 2,2′-dipyridyl disulphide (2-Py-S-S-2-Py) at pH4] per mol of protein is readily prepared both from dried papaya latex and from commercial 2×crystallized partially active papain. 4. The catalysis of the hydrolysis of α-N-benzoyl-l-arginine ethyl ester at pH6.0, 25.0°C, I=0.3 by fully active papain thus prepared is characterized by Km=18.2±<0.1mm and kcat.=16.4±0.5s−1. PMID:4733241

Structural and optical modification in 4H-SiC following 30 keV silver ion irradiation

NASA Astrophysics Data System (ADS)

Kaushik, Priya Darshni; Aziz, Anver; Siddiqui, Azher M.; Lakshmi, G. B. V. S.; Syväjärvi, Mikael; Yakimova, Rositsa; Yazdi, G. Reza

2018-05-01

The market of high power, high frequency and high temperature based electronic devices is captured by SiC due to its superior properties like high thermal conductivity and high sublimation temperature and also due to the limitation of silicon based electronics in this area. There is a need to investigate effect of ion irradiation on SiC due to its application in outer space as outer space is surrounded both by low and high energy ion irradiations. In this work, effect of low energy ion irradiation on structural and optical property of 4H-SiC is investigated. ATR-FTIR is used to study structural modification and UV-Visible spectroscopy is used to study optical modifications in 4H-SiC following 30 keV Ag ion irradiation. FTIR showed decrease in bond density of SiC along the ion path (track) due to the creation of point defects. UV-Visible absorption spectra showed decrease in optical band gap from 3.26 eV to 2.9 eV. The study showed degradation of SiC crystallity and change in optical band gap following low energy ion irradiation and should be addressed while fabricationg devices based on SiC for outer space application. Additionally, this study provides a platform for introducing structural and optical modification in 4H-SiC using ion beam technology in a controlled manner.

Enhanced enzyme stability through site-directed covalent immobilization.

PubMed

Wu, Jeffrey Chun Yu; Hutchings, Christopher Hayden; Lindsay, Mark Jeffrey; Werner, Christopher James; Bundy, Bradley Charles

2015-01-10

Breakthroughs in enzyme immobilization have enabled increased enzyme recovery and reusability, leading to significant decreases in the cost of enzyme use and fueling biocatalysis growth. However, current enzyme immobilization techniques suffer from leaching, enzyme stability, and recoverability and reusability issues. Moreover, these techniques lack the ability to control the orientation of the immobilized enzymes. To determine the impact of orientation on covalently immobilized enzyme activity and stability, we apply our PRECISE (Protein Residue-Explicit Covalent Immobilization for Stability Enhancement) system to a model enzyme, T4 lysozyme. The PRECISE system uses non-canonical amino acid incorporation and the Huisgen 1,3-dipolar cycloaddition "click" reaction to enable directed enzyme immobilization at rationally chosen residues throughout an enzyme. Unlike previous site-specific systems, the PRECISE system is a truly covalent immobilization method. Utilizing this system, enzymes immobilized at proximate and distant locations from the active site were tested for activity and stability under denaturing conditions. Our results demonstrate that orientation control of covalently immobilized enzymes can provide activity and stability benefits exceeding that of traditional random covalent immobilization techniques. PRECISE immobilized enzymes were 50 and 73% more active than randomly immobilized enzymes after harsh freeze-thaw and chemical denaturant treatments. Copyright © 2014 Elsevier B.V. All rights reserved.

Painting proteins with covalent labels: what's in the picture?

PubMed

Fitzgerald, Michael C; West, Graham M

2009-06-01

Knowledge about the structural and biophysical properties of proteins when they are free in solution and/or in complexes with other molecules is essential for understanding the biological processes that proteins regulate. Such knowledge is also important to drug discovery efforts, particularly those focused on the development of therapeutic agents with protein targets. In the last decade a variety of different covalent labeling techniques have been used in combination with mass spectrometry to probe the solution-phase structures and biophysical properties of proteins and protein-ligand complexes. Highlighted here are five different mass spectrometry-based covalent labeling strategies including: continuous hydrogen/deuterium (H/D) exchange labeling, hydroxyl radical-mediated footprinting, SUPREX (stability of unpurified proteins from rates of H/D exchange), PLIMSTEX (protein-ligand interaction by mass spectrometry, titration, and H/D exchange), and SPROX (stability of proteins from rates of oxidation). The basic experimental protocols used in each of the above-cited methods are summarized along with the kind of biophysical information they generate. Also discussed are the relative strengths and weaknesses of the different methods for probing the wide range of conformational states that proteins and protein-ligand complexes can adopt when they are in solution.

Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification

NASA Astrophysics Data System (ADS)

Tessarz, Peter; Santos-Rosa, Helena; Robson, Sam C.; Sylvestersen, Kathrine B.; Nelson, Christopher J.; Nielsen, Michael L.; Kouzarides, Tony

2014-01-01

Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (facilitator of chromatin transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 shows reduced histone incorporation and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.

Enhancer-associated H3K4 monomethylation by Trithorax-related, the Drosophila homolog of mammalian Mll3/Mll4.

PubMed

Herz, Hans-Martin; Mohan, Man; Garruss, Alexander S; Liang, Kaiwei; Takahashi, Yoh-Hei; Mickey, Kristen; Voets, Olaf; Verrijzer, C Peter; Shilatifard, Ali

2012-12-01

Monomethylation of histone H3 on Lys 4 (H3K4me1) and acetylation of histone H3 on Lys 27 (H3K27ac) are histone modifications that are highly enriched over the body of actively transcribed genes and on enhancers. Although in yeast all H3K4 methylation patterns, including H3K4me1, are implemented by Set1/COMPASS (complex of proteins associated with Set1), there are three classes of COMPASS-like complexes in Drosophila that could carry out H3K4me1 on enhancers: dSet1, Trithorax, and Trithorax-related (Trr). Here, we report that Trr, the Drosophila homolog of the mammalian Mll3/4 COMPASS-like complexes, can function as a major H3K4 monomethyltransferase on enhancers in vivo. Loss of Trr results in a global decrease of H3K4me1 and H3K27ac levels in various tissues. Assays with the cut wing margin enhancer implied a functional role for Trr in enhancer-mediated processes. A genome-wide analysis demonstrated that Trr is required to maintain the H3K4me1 and H3K27ac chromatin signature that resembles the histone modification patterns described for enhancers. Furthermore, studies in the mammalian system suggested a role for the Trr homolog Mll3 in similar processes. Since Trr and mammalian Mll3/4 complexes are distinguished by bearing a unique subunit, the H3K27 demethylase UTX, we propose a model in which the H3K4 monomethyltransferases Trr/Mll3/Mll4 and the H3K27 demethylase UTX cooperate to regulate the transition from inactive/poised to active enhancers.

Enhancer-associated H3K4 monomethylation by Trithorax-related, the Drosophila homolog of mammalian Mll3/Mll4

PubMed Central

Herz, Hans-Martin; Mohan, Man; Garruss, Alexander S.; Liang, Kaiwei; Takahashi, Yoh-hei; Mickey, Kristen; Voets, Olaf; Verrijzer, C. Peter; Shilatifard, Ali

2012-01-01

Monomethylation of histone H3 on Lys 4 (H3K4me1) and acetylation of histone H3 on Lys 27 (H3K27ac) are histone modifications that are highly enriched over the body of actively transcribed genes and on enhancers. Although in yeast all H3K4 methylation patterns, including H3K4me1, are implemented by Set1/COMPASS (complex of proteins associated with Set1), there are three classes of COMPASS-like complexes in Drosophila that could carry out H3K4me1 on enhancers: dSet1, Trithorax, and Trithorax-related (Trr). Here, we report that Trr, the Drosophila homolog of the mammalian Mll3/4 COMPASS-like complexes, can function as a major H3K4 monomethyltransferase on enhancers in vivo. Loss of Trr results in a global decrease of H3K4me1 and H3K27ac levels in various tissues. Assays with the cut wing margin enhancer implied a functional role for Trr in enhancer-mediated processes. A genome-wide analysis demonstrated that Trr is required to maintain the H3K4me1 and H3K27ac chromatin signature that resembles the histone modification patterns described for enhancers. Furthermore, studies in the mammalian system suggested a role for the Trr homolog Mll3 in similar processes. Since Trr and mammalian Mll3/4 complexes are distinguished by bearing a unique subunit, the H3K27 demethylase UTX, we propose a model in which the H3K4 monomethyltransferases Trr/Mll3/Mll4 and the H3K27 demethylase UTX cooperate to regulate the transition from inactive/poised to active enhancers. PMID:23166019

A hydroxyapatite coating covalently linked onto a silicone implant material.

PubMed

Furuzono, T; Sonoda, K; Tanaka, J

2001-07-01

A novel composite consisting of hydroxyapatite (HAp) microparticles covalently coupled onto a silicone sheet was developed. Initially, an acrylic acid (AAc) -grafted silicone sheet with a 16.7 microg/cm(2) surface graft density was prepared by corona-discharge treatment. The surface of sintered, spherical, carbonated HAp particles with an average diameter of 2.0 microm was subsequently modified with amino groups. The amino group surface density of the HAp particles was calculated to be approximately one amino molecule per 1.0 nm(2) of particle surface area. These samples were characterized with Fourier transform infrared spectrometry and X-ray photoelectron spectroscopy. After the formation of ammonium ionic bonds between both samples under aqueous conditions, they were reacted at 180 degrees C for 6 h in vacuo to form covalent bonds through a solid-phase condensation. The HAp particles were coupled to the AAc-grafted silicone surface by a covalent linkage. Further improvements in the adhesive and bioactive properties of the HAp-coated silicone material are expected.

The small molecule 2-phenylethynesulfonamide induces covalent modification of p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jamil, Sarwat; Hojabrpour, Payman; Duronio, Vincent

p53 is a tumor suppressor protein which is either lost or inactivated in a large majority of tumors. The small molecule 2-phenylethynesulfonamide (PES) was originally identified as the inhibitor of p53 effects on the mitochondrial death pathway. In this report we demonstrate that p53 protein from PES-treated cells was detected in reduced mobility bands between molecular weights 95–220 kDa. Resolution of p53 aggregates on urea gel was unable to reduce the high molecular weight p53 aggregates, which were shown to be primarily located in the nucleus. Therefore, our data suggest that PES exerts its effects through covalent cross-linking and nuclear retentionmore » of p53. - Highlights: • p53 protein is in high molecular weight complexes in the nucleus of PES-treated cells. • PES is a drug that inhibits pro-apoptotic p53 action at the mitochondria. • We propose that PES action involves cross-linking and nuclear retention of p53.« less

Molecular dynamics simulation on HP1 protein binding by histone H3 tail methylation and phosphorylation

NASA Astrophysics Data System (ADS)

Jiang, Yan-Ke; Zou, Jian-Wei; Wu, Yu-Qian; Zhang, Na; Yu, Qing-Sen; Jiang, Yong-Jun

Trimethylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging, and heterochromatin formation. Phosphorylation of histone H3 has been linked with mitotic chromatin condensation. During mitosis in vivo, H3 lysine 9 methylation and serine 10 phosphorylation can occur concomitantly on the same histone tail, whereas the influence of phosphorylation to trimethylation H3 tail recruiting HP1 remains controversial. In this work, molecular dynamics simulation of HP1 complexed with both trimethylated and phosphorylated H3 tail were performed and compared with the results from the previous methylated H3-HP1 trajectory. It is clear from the 10-ns dynamics simulation that two adjacent posttranslational modifications directly increase the flexibility of the H3 tail and weaken HP1 binding to chromatin. A combinatorial readout of two adjacent posttranslational modifications-a stable methylation and a dynamic phosphorylation mark-establish a regulatory mechanism of protein-protein interactions.

Rare earth (Eu{sup 3+}, Tb{sup 3+}) mesoporous hybrids with calix[4]arene derivative covalently linking MCM-41: Physical characterization and photoluminescence property

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Yajuan; Yan Bing, E-mail: byan@tongji.edu.cn; Wang Li

2011-09-15

MCM-41 mesoporous silica has been functionalized with two kinds of macrocylic calixarene derivatives Calix[4] and Calix[4]Br (Calix[4]=P-tert-butylcalix[4]arene, Calix[4]Br=5.11,17.23-tetra-tert-butyl-25.27-bihydroxy- 26.28-bibromopropoxycalix[4]arene) through condensation approach of tetraethoxysilane (TEOS) in the presence of the cetyltrimethylammonium bromide (CTAB) surfactant as a template. Novel organic-inorganic mesoporous luminescent hybrid containing RE{sup 3+} (Eu{sup 3+}, Tb{sup 3+}) complexes covalently attached to the functionalized ordered mesoporous MCM-41, which are designated as RE-Calix[4]-MCM-41 and RE-Calix[4]Br-MCM-41, respectively, are obtained by sol-gel process. It is found that they all have high surface area, uniform in the mesostructure and good crystallinity. Measurement of the photoluminescence properties show the mesoporous material covalently bonded Tb{supmore » 3+} complexes (Tb-Calix[4]-MCM-41 and Tb-Calix[4]Br-MCM-41) exhibit the stronger characteristic emission of Tb{sup 3+} and longer lifetime than the corresponding Eu-containing materials Eu-Calix[4]-MCM-41 and Eu-Calix[4]Br-MCM-41 due to the triplet state energy of modified organic ligands Calix[4]-Si and Calix[4]Br-Si match with the emissive energy level of Tb{sup 3+} very well. - Graphical abstract: MCM-41 mesoporous silica is functionalized with two kinds of macrocylic calixarene derivatives and luminescent organic-inorganic mesoporous hybrids containing Ln{sup 3+} complexes covalently attached to the functionalized ordered mesoporous MCM-41. Highlights: > Novel linkages of functionalized calixarene derivative. > New rare earth mesoporous hybrids. > Luminescence in visible region.« less

The occurence of uric acids and the growth morphology of the anhydrous monoclinic modification : C 5H 4N 4O 3

NASA Astrophysics Data System (ADS)

Rinaudo, C.; Boistelle, R.

1980-07-01

Anhydrous and hydrated uric acid crystals which frequently occur in human urolithiasis have been grown from pure aqueous solutions at 25 and 37°C. The appearance domain of either modification is given as a function of initial uric acid concentration at pH values ranging from 3.0 to 5.5. The present paper mostly deals with the anhydrous phase and it is shown that its experimental and theoretical growth morphologies are in agreement, the crystals exhibiting the {100}, {210}, {121}, {001} and {201} forms. Crystal habit depends on supersaturation; {100} has always the largest extension at low supersaturation {121} develops to the detriment of {210}, {001} and {201}.

Installation of a reactive site for covalent wiring onto an intrinsically conductive poly(ionic liquid)

DOE PAGES

Brombosz, Scott M.; Lee, Sungwon; Firestone, Millicent A.

2014-11-04

We describe post-polymerization radical bromination of a nanostructured poly(ionic liquid) that selectively introduces a reactive bromo-group onto the polyalkylthiophene backbone. Raman and FT-IR spectroscopy proves that the bromine is successfully introduced at the 3-methyl position of the thiophene and that the molecular structure of the polymer remains largely intact with only minimal chain scission detected. FT-IR and Vis-NIR spectroscopy indicates that incorporation of the bromine induces twisting (loss of co-planarity) of the polythiophene backbone. WAXS confirms retention of an ordered lamellar structure with minor lattice spacing contraction. Cyclic voltammetry confirms spectroscopic findings that the bromination reaction yields a stable p-dopedmore » polymer. The installed bromine is susceptible to nucleophilic displacement permitting the covalent attachment of other functional molecules, such as a dialkylphosphonate. Elemental analysis of such a transformation established that 100 % functionalization can be achieved. These results collectively demonstrate that post-modification of a π-conjugated polymer can be used to both tune electronic and photonic properties, as well as install a chemoselective attachment point for the covalent wiring of other molecules.« less

Covalent dimers of 1,3-diphenylisobenzofuran for singlet fission: synthesis and electrochemistry.

PubMed

Akdag, Akin; Wahab, Abdul; Beran, Pavel; Rulíšek, Lubomír; Dron, Paul I; Ludvík, Jiří; Michl, Josef

2015-01-02

The synthesis of covalent dimers in which two 1,3-diphenylisobenzofuran units are connected through one phenyl substituent on each is reported. In three of the dimers, the subunits are linked directly, and in three others, they are linked via an alkane chain. A seventh new compound in which two 1,3-diphenylisobenzofuran units share a phenyl substituent is also described. These materials are needed for investigations of the singlet fission process, which promises to increase the efficiency of solar cells. The electrochemical oxidation and reduction of the monomer, two previously known dimers, and the seven new compounds have been examined, and reversible redox potentials have been compared with results obtained from density functional theory. Although the overall agreement is satisfactory, some discrepancies are noted and discussed.

Characterization of Covalent-Reversible EGFR Inhibitors

PubMed Central

2017-01-01

Within the spectrum of kinase inhibitors, covalent-reversible inhibitors (CRIs) provide a valuable alternative approach to classical covalent inhibitors. This special class of inhibitors can be optimized for an extended drug-target residence time. For CRIs, it was shown that the fast addition of thiols to electron-deficient olefins leads to a covalent bond that can break reversibly under proteolytic conditions. Research groups are just beginning to include CRIs in their arsenal of compound classes, and, with that, the understanding of this interesting set of chemical warheads is growing. However, systems to assess both characteristics of the covalent-reversible bond in a simple experimental setting are sparse. Here, we have developed an efficient methodology to characterize the covalent and reversible properties of CRIs and to investigate their potential in targeting clinically relevant variants of the receptor tyrosine kinase EGFR.

Histone modifications associated with both A and B chromosomes of maize.

PubMed

Jin, Weiwei; Lamb, Jonathan C; Zhang, Wenli; Kolano, Bozena; Birchler, James A; Jiang, Jiming

2008-01-01

We report the distribution of several histone modifications along the arms and in centromeric regions of somatic chromosomes of maize, including the supernumerary B chromosome. Acetylated H3 and H4 as well as H3K4me2, modifications associated with euchromatin, were enriched in the distal parts of the A chromosomes, but were progressively depleted toward the centromeres of the A chromosomes and were depleted in the heterochromatic portions of the B chromosome. Classical histone modifications associated with heterochromatin, including H3K9me2, H3K27me1 and H3K27me2, were distributed throughout both A and B chromosomes. However, H3K27me2 showed a reduced level on the B chromosome compared with the A chromosomes and was not associated with some classes of constitutive heterochromatin. We monitored the presence of each histone modification in the centromeric regions using a YFP-tagged centromere-specific histone, CENH3. We observed the presence of H3K9me2 and absence of H3K4me2 in the centromeric regions of both A and B chromosomes of maize, which is in contrast to the presence of H3K4me2 and absence of H3K9me2 in animal centromeres. These results show a diversity of epigenetic modifications associated with centromeric chromatin in different eukaryotes.

Association of H3K9me3 and H3K27me3 repressive histone marks with breast cancer subtypes in the Nurses' Health Study.

PubMed

Healey, Megan A; Hu, Rong; Beck, Andrew H; Collins, Laura C; Schnitt, Stuart J; Tamimi, Rulla M; Hazra, Aditi

2014-10-01

Repressive histone tail modifications have been associated with molecular breast cancer subtypes. We investigated whether histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) were associated with tumor features and subtypes while adjusting for prospectively collected reproductive and lifestyle breast cancer risk factors. We have tissue microarray data with immunohistochemical marker information on 804 incident cases of invasive breast cancer diagnosed from 1976-2000 in the Nurses' Health Study. Tissue microarray sections were stained for global H3K9me3 and H3K27me3, and scored into four categories. Multivariate odds ratios (OR) and 95 % confidence intervals (CI) were calculated using logistic regression models for tumor features and subtypes, adjusting for breast cancer risk factors. While there were no significant associations between H3K9me3 and tumor features, H3K27me3 was significantly associated with lower grade tumors compared to high grade tumors in the multivariate model (OR = 1.95, 95 % CI 1.35-2.81, p = 0.0004). H3K27me3 was suggestively associated with estrogen receptor-positive (ER+) tumors (OR = 1.47, 95 % CI 0.97-2.23, p = 0.07). In subtype analyses, H3K27me3 was positively associated with the luminal A subtype compared to all other subtypes (OR = 1.42, 95 % CI 1.14-1.77, p = 0.002), and was inversely associated with HER2-type (OR = 0.58, 95 % CI 0.37-0.91, p = 0.02) and basal-like breast cancer (OR = 0.52, 95 % CI 0.36-0.76, p = 0.0006). In the largest immunohistochemical examination of H3K9me3 and H3K27me3 in breast cancer, we found that H3K27me3 positivity, but not H3K9me3, was associated with lower grade tumors and the luminal A subtype after adjusting for reproductive and lifestyle breast cancer risk factors.

Lignin Modification for Biopolymer/Conjugated Polymer Hybrids as Renewable Energy Storage Materials.

PubMed

Nilsson, Ting Yang; Wagner, Michal; Inganäs, Olle

2015-12-07

Lignin derivatives, which arise as waste products from the pulp and paper industry and are mainly used for heating, can be used as charge storage materials. The charge storage function is a result of the quinone groups formed in the lignin derivative. Herein, we modified lignins to enhance the density of such quinone groups by covalently linking monolignols and quinones through phenolation. The extra guaiacyl, syringyl, and hydroquinone groups introduced by phenolation of kraft lignin derivatives were monitored by (31) P nuclear magnetic resonance and size exclusion chromatography. Electropolymerization in ethylene glycol/tetraethylammonium tosylate electrolyte was used to synthesize the kraft lignin/polypyrrole hybrid films. These modifications changed the phenolic content of the kraft lignin with attachment of hydroquinone units yielding the highest specific capacity (around 70 mA h g(-1) ). The modification of softwood and hardwood lignin derivatives yielded 50 % and 23 % higher charge capacity than the original lignin, respectively. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

X-ray diffraction analysis of 4- and 4'-substituted C n H2 n + 1O-C6H3(OH)-CH=N-C6H4-C m H2 m + 1 ( n/ m = 2/1 and 3/4) salicylideneanilines

NASA Astrophysics Data System (ADS)

Kuz'mina, L. G.; Navasardyan, M. A.; Mikhailov, A. A.

2017-11-01

X-ray diffraction study of two crystalline modifications of C2H5O-C6H3(OH)-CH=N-C6H4-CH3 ( 1a, sp. gr. P21/ n, and 1b, sp. gr. C2/c) and C3H7O-C6H3(OH)-CH=N-C6H4-C4H9 ( 2, sp. gr. P212121) has been performed. The 1a crystal structure contains two independent molecules. The molecules are conformationally nonrigid with respect to the mutual rotation of benzene rings; the dihedral angles between their planes are 29.19° and 26.00° in the independent molecules of 1a, 18.72° in the molecule of 1b, and 50.35° in the molecule of 2. The crystal packing of the compounds is discussed.

Age-related macular degeneration and modification of systemic complement factor H production through liver transplantation.

PubMed

Khandhadia, Samir; Hakobyan, Svetlana; Heng, Ling Z; Gibson, Jane; Adams, David H; Alexander, Graeme J; Gibson, Jonathan M; Martin, Keith R; Menon, Geeta; Nash, Kathryn; Sivaprasad, Sobha; Ennis, Sarah; Cree, Angela J; Morgan, B Paul; Lotery, Andrew J

2013-08-01

To investigate whether modification of liver complement factor H (CFH) production, by alteration of liver CFH Y402H genotype through liver transplantation (LT), influences the development of age-related macular degeneration (AMD). Multicenter, cross-sectional study. We recruited 223 Western European patients ≥ 55 years old who had undergone LT ≥ 5 years previously. We determined AMD status using a standard grading system. Recipient CFH Y402H genotype was obtained from DNA extracted from recipient blood samples. Donor CFH Y402H genotype was inferred from recipient plasma CFH Y402H protein allotype, measured using enzyme-linked immunosorbent assays. This approach was verified by genotyping donor tissue from a subgroup of patients. Systemic complement activity was ascertained by measuring levels of plasma complement proteins using an enzyme-linked immunosorbent assay, including substrates (C3, C4), activation products (C3a, C4a, and terminal complement complex), and regulators (total CFH, C1 inhibitor). We evaluated AMD status and recipient and donor CFH Y402H genotype. In LT patients, AMD was associated with recipient CFH Y402H genotype (P = 0.036; odds ratio [OR], 1.6; 95% confidence interval [CI], 1.0-2.4) but not with donor CFH Y402H genotype (P = 0.626), after controlling for age, sex, smoking status, and body mass index. Recipient plasma CFH Y402H protein allotype predicted donor CFH Y402H genotype with 100% accuracy (n = 49). Plasma complement protein or activation product levels were similar in LT patients with and without AMD. Compared with previously reported prevalence figures (Rotterdam Study), LT patients demonstrated a high prevalence of both AMD (64.6% vs 37.1%; OR, 3.09; P<0.001) and the CFH Y402H sequence variation (41.9% vs 36.2%; OR, 1.27; P = 0.014). Presence of AMD is not associated with modification of hepatic CFH production. In addition, AMD is not associated with systemic complement activity in LT patients. These findings suggest that local

A histone H3K36 chromatin switch coordinates DNA double-strand break repair pathway choice.

PubMed

Pai, Chen-Chun; Deegan, Rachel S; Subramanian, Lakxmi; Gal, Csenge; Sarkar, Sovan; Blaikley, Elizabeth J; Walker, Carol; Hulme, Lydia; Bernhard, Eric; Codlin, Sandra; Bähler, Jürg; Allshire, Robin; Whitehall, Simon; Humphrey, Timothy C

2014-06-09

DNA double-strand break (DSB) repair is a highly regulated process performed predominantly by non-homologous end joining (NHEJ) or homologous recombination (HR) pathways. How these pathways are coordinated in the context of chromatin is unclear. Here we uncover a role for histone H3K36 modification in regulating DSB repair pathway choice in fission yeast. We find Set2-dependent H3K36 methylation reduces chromatin accessibility, reduces resection and promotes NHEJ, while antagonistic Gcn5-dependent H3K36 acetylation increases chromatin accessibility, increases resection and promotes HR. Accordingly, loss of Set2 increases H3K36Ac, chromatin accessibility and resection, while Gcn5 loss results in the opposite phenotypes following DSB induction. Further, H3K36 modification is cell cycle regulated with Set2-dependent H3K36 methylation peaking in G1 when NHEJ occurs, while Gcn5-dependent H3K36 acetylation peaks in S/G2 when HR prevails. These findings support an H3K36 chromatin switch in regulating DSB repair pathway choice.

Thermoelectric properties of the 3C, 2H, 4H, and 6H polytypes of the wide-band-gap semiconductors SiC, GaN, and ZnO

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Zheng; Lü, Tie-Yu; Wang, Hui-Qiong

We have investigated the thermoelectric properties of the 3C, 2H, 4H, and 6H polytypes of the wide-band-gap(n-type) semiconductors SiC, GaN, and ZnO based on first-principles calculations and Boltzmann transport theory. Our results show that the thermoelectric performance increases from 3C to 6H, 4H, and 2H structures with an increase of hexagonality for SiC. However, for GaN and ZnO, their power factors show a very weak dependence on the polytype. Detailed analysis of the thermoelectric properties with respect to temperature and carrier concentration of 4H-SiC, 2H-GaN, and 2H-ZnO shows that the figure of merit of these three compounds increases with temperature,more » indicating the promising potential applications of these thermoelectric materials at high temperature. The significant difference of the polytype-dependent thermoelectric properties among SiC, GaN, and ZnO might be related to the competition between covalency and ionicity in these semiconductors. Our calculations may provide a new way to enhance the thermoelectric properties of wide-band-gap semiconductors through atomic structure design, especially hexagonality design for SiC.« less

Photoactivable antibody binding protein: site-selective and covalent coupling of antibody.

PubMed

Jung, Yongwon; Lee, Jeong Min; Kim, Jung-won; Yoon, Jeongwon; Cho, Hyunmin; Chung, Bong Hyun

2009-02-01

Here we report new photoactivable antibody binding proteins, which site-selectively capture antibodies and form covalent conjugates with captured antibodies upon irradiation. The proteins allow the site-selective tagging and/or immobilization of antibodies with a highly preferred orientation and omit the need for prior antibody modifications. The minimal Fc-binding domain of protein G, a widely used antibody binding protein, was genetically and chemically engineered to contain a site-specific photo cross-linker, benzophenone. In addition, the domain was further mutated to have an enhanced Fc-targeting ability. This small engineered protein was successfully cross-linked only to the Fc region of the antibody without any nonspecific reactivity. SPR analysis indicated that antibodies can be site-selectively biotinylated through the present photoactivable protein. Furthermore, the system enabled light-induced covalent immobilization of antibodies directly on various solid surfaces, such as those of glass slides, gold chips, and small particles. Antibody coupling via photoactivable antibody binding proteins overcomes several limitations of conventional approaches, such as random chemical reactions or reversible protein binding, and offers a versatile tool for the field of immunosensors.

Association rate constants for reactions between resonance-stabilized radicals: C 3H 3 + C 3H 3, C 3H 3 + C 3H 5, and C 3H 5 + C 3H 5

DOE PAGES

Georgievskii, Yuri; Miller, James A.; Klippenstein, Stephen J.

2007-05-18

Reactions between resonance-stabilized radicals play an important role in combustion chemistry. The theoretical prediction of rate coefficients and product distributions for such reactions is complicated by the fact that the initial complex-formation steps and some dissociation steps are barrierless. In this work, direct variable reaction coordinate transition state theory (VRC-TST) is used to predict accurately the association rate constants for the self and cross reactions of propargyl and allyl radicals. For each reaction, a set of multifaceted dividing surfaces is used to account for the multiple possible addition channels. Because of their resonant nature the geometric relaxation of the radicalsmore » is important. Here, the effect of this relaxation is explicitly calculated with the UB3LYP/cc-pvdz method for each mutual orientation encountered in the configurational integrals over the transition state dividing surfaces. The final energies are obtained from CASPT2/cc-pvdz calculations with all π-orbitals in the active space. Evaluations along the minimum energy path suggest that basis set corrections are negligible. The VRC-TST approach was also used to calculate the association rate constant and the corresponding number of states for the C 6H 5 + H → C 6H 6 exit channel of the C 3H 3 + C 3H 3 reaction, which is also barrierless. For this reaction, the interaction energies were evaluated with the CASPT2(2e,2o)/cc-pvdz method and a 1-D correction is included on the basis of CAS+1+2+QC/aug-cc-pvtz calculations for the CH 3 + H reference system. For the C 3H 3 + C 3H 3 reaction, the VRC-TST results for the energy and angular momentum resolved numbers of states in the entrance channels and in the C 6H 5 + H exit channel are incorporated in a master equation simulation to determine the temperature and pressure dependence of the phenomenological rate coefficients. The rate constants for the C 3H 3 + C 3H 3 and C 3H 5 + C 3H 5 self-reactions compare favorably

Succinimidyl Ester Surface Chemistry: Implications of the Competition between Aminolysis and Hydrolysis on Covalent Protein Immobilization

PubMed Central

2015-01-01

N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent protein immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to protein immobilization suggests that under coupling conditions in which low protein concentrations and buffers of near physiological pH are used, proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins. PMID:25317495

Chemical modification and pH dependence of kinetic parameters to identify functional groups in a glucosyltransferase from Strep. Mutans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bell, J.E.; Leone, A.; Bell, E.T.

1986-05-01

A glucosyltransferase, forming a predominantly al-6 linked glucan, was partially purified from the culture filtrate of S. mutans GS-5. The kinetic properties of the enzyme, assessed using the transfer of /sup 14/C glucose from sucrose into total glucan, were studied at pH values from pH 3.5 to 6.5. From the dependence of km on pH, a group with pKa = 5.5 must be protonated to maximize substrate binding. From plots of V/sub max/ vs pH two groups, with pKa's of 4.5 and 5.5 were indicated. The results suggest the involvement of either two carboxyl groups (one protonated, one unprotonated inmore » the native enzyme) or a carboxyl group (unprotonated) and some other protonated group such as histidine, cysteine. Chemical modification studies showed that Diethylyrocarbonate (histidine specific) had no effect on enzyme activity while modification with p-phydroxy-mercuribenzoate or iodoacetic acid (sulfhydryl reactive) and carbodimide reagents (carboxyl specific) resulted in almost complete inactivation. Activity loss was dependent upon time of incubation and reagent concentration. The disaccharide lylose, (shown to be an inhibitor of the enzyme with similar affinity to sucrose) offers no protection against modification by the sulfhydryl reactive reagents.« less

Covalent Functionalization of NiTi Surfaces with Bioactive Peptide Amphiphile Nanofibers

PubMed Central

Sargeant, Timothy D.; Rao, Mukti S.; Koh, Chung-Yan

2009-01-01

Surface modification enables the creation of bioactive implants using traditional material substrates without altering the mechanical properties of the bulk material. For applications such as bone plates and stents, it is desirable to modify the surface of metal alloy substrates to facilitate cellular attachment, proliferation, and possibly differentiation. In this work we present a general strategy for altering the surface chemistry of nickel-titanium shape memory alloy (NiTi) in order to covalently attach self-assembled peptide amphiphile (PA) nanofibers with bioactive functions. Bioactivity in the systems studied here includes biological adhesion and proliferation of osteoblast and endothelial cell types. The optimized surface treatment creates a uniform TiO2 layer with low levels of Ni on the NiTi surface, which is subsequently covered with an aminopropylsilane coating using a novel, lower temperature vapor deposition method. This method produces an aminated surface suitable for covalent attachment of PA molecules containing terminal carboxylic acid groups. The functionalized NiTi surfaces have been characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and atomic force microscopy (AFM). These techniques offer evidence that the treated metal surfaces consist primarily of TiO2 with very little Ni, and also confirm the presence of the aminopropylsilane overlayer. Self-assembled PA nanofibers presenting the biological peptide adhesion sequence Arg-Gly-Asp-Ser are capable of covalently anchoring to the treated substrate, as demonstrated by spectrofluorimetry and AFM. Cell culture and scanning electron microscopy (SEM) demonstrate cellular adhesion, spreading, and proliferation on these functionalized metal surfaces. Furthermore, these experiments demonstrate that covalent attachment is crucial for creating robust PA nanofiber coatings, leading to confluent cell monolayers. PMID:18083225

Chemical Modification of Papain and Subtilisin: An Active Site Comparison

ERIC Educational Resources Information Center

St-Vincent, Mireille; Dickman, Michael

2004-01-01

An experiment using methyle methanethiosulfonate (MMTS) and phenylmethylsulfonyl flouride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilism respectively is demonstrated. The covalent modification of these enzymes and subsequent rescue of papain shows the beginning biochemist that proteins…

Modification of silicon nitride surfaces with GOPES and APTES for antibody immobilization: computational and experimental studies

NASA Astrophysics Data System (ADS)

Dien To, Thien; Nguyen, Anh Tuan; Nhat Thanh Phan, Khoa; Thu Thi Truong, An; Doan, Tin Chanh Duc; Mau Dang, Chien

2015-12-01

Chemical modification of silicon nitride (SiN) surfaces by silanization has been widely studied especially with 3-(aminopropyl)triethoxysilane (APTES) and 3-(glycidyloxypropyl) dimethylethoxysilane (GOPES). However few reports performed the experimental and computational studies together. In this study, surface modification of SiN surfaces with GOPES and APTES covalently bound with glutaraldehyde (GTA) was investigated for antibody immobilization. The monoclonal anti-cytokeratin-FITC (MACF) antibody was immobilized on the modified SiN surfaces. The modified surfaces were characterized by water contact angle measurements, atomic force microscopy and fluorescence microscopy. The FITC-fluorescent label indicated the existence of MACF antibody on the SiN surfaces and the efficiency of the silanization reaction. Absorption of APTES and GOPES on the oxidized SiN surfaces was computationally modeled and calculated by Materials Studio software. The computational and experimental results showed that modification of the SiN surfaces with APTES and GTA was more effective than the modification with GOPES.

Unimolecular Micelles of Amphiphilic Cyclodextrin-Core Star-Like Copolymers with Covalent pH-Responsive Linkage of Anticancer Prodrugs.

PubMed

Jia, Tao; Huang, Shuo; Yang, Cangjie; Wang, Mingfeng

2017-08-07

Multifunctional stable and stimuli-responsive drug delivery systems are important for efficient cancer treatment due to their advantages such as enhanced cancer-targeting efficiency, improved pharmacokinetics, minimized drug leaching, and reduced undesirable side effects. Here we report a robust and pH-responsive anticancer drug delivery system based on unimolecular micelles of star-like amphiphilic copolymers. The polymers (denoted as CPOFs) were facilely synthesized via one-step atom transfer radical polymerization of functionalizable benzoaldehyde and hydrophilic poly[(oligo ethylene glycol) methyl ether methacrylate] as comonomers from the core of heptakis [2,3,6-tri-o-(2-bromo-2-methyl propionyl]-β-cyclodextrin as the initiator. Doxorubicin (DOX) as an anticancer drug was covalently linked to the benzoaldehyde groups of CPOFs through pH-sensitive Schiff-base bonds. The DOX-conjugated polymers, denoted as CPOF-DOX, formed robust unimolecular micelles with an average diameter of 18 nm in aqueous media. More importantly, these unimolecular micelles showed higher drug loading capacity and more controllable drug release characteristics, compared to our previous unimolecular micelles of β-cyclodextrin-poly(lactic acid)-b-poly[(oligo ethylene glycol) methyl ether methacrylates] that physically encapsulated DOX via hydrophobic interaction. Moreover, the CPOF-DOX unimolecular micelles could be internalized by human cervical cancer HeLa cells in a stepwise way and showed less cytotoxicity compared to carrier-free DOX. We foresee that CPOF-DOX would provide a promising robust and controllable anticancer drug delivery system for future animal study and clinical trials for cancer treatment.

Functionalization of multiwalled carbon nanotubes by microwave irradiation for lysozyme attachment: comparison of covalent and adsorption methods by kinetics of thermal inactivation

NASA Astrophysics Data System (ADS)

Puentes-Camacho, Daniel; Velázquez, Enrique F.; Rodríguez-Félix, Dora E.; Castillo-Ortega, Mónica; Sotelo-Mundo, Rogerio R.; del Castillo-Castro, Teresa

2017-12-01

Proteins suffer changes in their tertiary structure when they are immobilized, and enzymatic activity is affected due to the low biocompatibility of some supporting materials. In this work immobilization of lysozyme on carbon nanotubes previously functionalized by microwave irradiation was studied. The effectiveness of the microwave-assisted acid treatment of carbon nanotubes was evaluated by XPS, TEM, Raman and FTIR spectroscopy. The carboxylic modification of nanotube surfaces by this fast, simple and feasible method allowed the physical adsorption and covalent linking of active lysozyme onto the carbonaceous material. Thermal inactivation kinetics, thermodynamic parameters and storage stability were studied for adsorbed and covalent enzyme complexes. A major stability was found for lysozyme immobilized by the covalent method, the activation energy for inactivation of the enzyme was higher for the covalent method and it was stable after 50 d of storage at 4 °C. The current study highlights the effect of protein immobilization method on the biotechnological potential of nanostructured biocatalysts.

Formation of Covalently Modified Folding Intermediates of Simian Virus 40 Vp1 in Large T Antigen-Expressing Cells

PubMed Central

Watanabe, Marika; Phamduong, Ellen; Huang, Chu-Han; Itoh, Noriko; Bernal, Janie; Nakanishi, Akira; Rundell, Kathleen; Gjoerup, Ole

2013-01-01

The folding and pentamer assembly of the simian virus 40 (SV40) major capsid protein Vp1, which take place in the infected cytoplasm, have been shown to progress through disulfide-bonded Vp1 folding intermediates. In this report, we further demonstrate the existence of another category of Vp1 folding or assembly intermediates: the nonreducible, covalently modified mdVp1s. These species were present in COS-7 cells that expressed a recombinant SV40 Vp1, Vp1ΔC, through plasmid transfection. The mdVp1s persisted under cell and lysate treatment and SDS-PAGE conditions that are expected to have suppressed the formation of artifactual disulfide cross-links. As shown through a pulse-chase analysis, the mdVp1s were derived from the newly synthesized Vp1ΔC in the same time frame as Vp1's folding and oligomerization. The apparent covalent modifications occurred in the cytoplasm within the core region of Vp1 and depended on the coexpression of the SV40 large T antigen (LT) in the cells. Analogous covalently modified species were found with the expression of recombinant polyomavirus Vp1s and human papillomavirus L1s in COS-7 cells. Furthermore, the mdVp1s formed multiprotein complexes with LT, Hsp70, and Hsp40, and a fraction of the largest mdVp1, md4, was disulfide linked to the unmodified Vp1ΔC. Both mdVp1 formation and most of the multiprotein complex formation were blocked by a Vp1 folding mutation, C87A-C254A. Our observations are consistent with a role for LT in facilitating the folding process of SV40 Vp1 by stimulating certain covalent modifications of Vp1 or by recruiting certain cellular proteins. PMID:23427157

Relevant insight of surface characterization techniques to study covalent grafting of a biopolymer to titanium implant and its acidic resistance

NASA Astrophysics Data System (ADS)

D'Almeida, Mélanie; Amalric, Julien; Brunon, Céline; Grosgogeat, Brigitte; Toury, Bérangère

2015-02-01

Peri-implant bacterial infections are the main cause of complications in dentistry. Our group has previously proposed the attachment of chitosan on titanium implants via a covalent bond to improve its antibacterial properties while maintaining its biocompatibility. A better knowledge of the coating preparation process allows a better understanding of the bioactive coating in biological conditions. In this work, several relevant characterization techniques were used to assess an implant device during its production phase and its resistance in natural media at different pH. The titanium surface was functionalized with 3-aminopropyltriethoxysilane (APTES) followed by grafting of an organic coupling agent; succinic anhydride, able to form two covalent links, with the substrate through a Ti-O-Si bond and the biopolymer through a peptide bond. Each step of the coating synthesis as well as the presence confirmation of the biopolymer on titanium after saliva immersion was followed by FTIR-ATR, SEM, EDS, 3D profilometry, XPS and ToF-SIMS analyses. Results allowed to highlight the efficiency of each step of the process, and to propose a mechanism occurring during the chitosan coating degradation in saliva media at pH 5 and at pH 3.

H3K4me3 breadth is linked to cell identity and transcriptional consistency.

PubMed

Benayoun, Bérénice A; Pollina, Elizabeth A; Ucar, Duygu; Mahmoudi, Salah; Karra, Kalpana; Wong, Edith D; Devarajan, Keerthana; Daugherty, Aaron C; Kundaje, Anshul B; Mancini, Elena; Hitz, Benjamin C; Gupta, Rakhi; Rando, Thomas A; Baker, Julie C; Snyder, Michael P; Cherry, J Michael; Brunet, Anne

2014-07-31

Trimethylation of histone H3 at lysine 4 (H3K4me3) is a chromatin modification known to mark the transcription start sites of active genes. Here, we show that H3K4me3 domains that spread more broadly over genes in a given cell type preferentially mark genes that are essential for the identity and function of that cell type. Using the broadest H3K4me3 domains as a discovery tool in neural progenitor cells, we identify novel regulators of these cells. Machine learning models reveal that the broadest H3K4me3 domains represent a distinct entity, characterized by increased marks of elongation. The broadest H3K4me3 domains also have more paused polymerase at their promoters, suggesting a unique transcriptional output. Indeed, genes marked by the broadest H3K4me3 domains exhibit enhanced transcriptional consistency and [corrected] increased transcriptional levels, and perturbation of H3K4me3 breadth leads to changes in transcriptional consistency. Thus, H3K4me3 breadth contains information that could ensure transcriptional precision at key cell identity/function genes. Copyright © 2014 Elsevier Inc. All rights reserved.

Computational design of soft materials for the capture of Cs-137 in contaminated environments: From 2D covalent cucurbituril networks to 3D supramolecular materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pichierri, Fabio, E-mail: fabio@che.tohoku.ac.jp

Using computational quantum chemistry methods we design novel 2D and 3D soft materials made of cucurbituril macrocycles covalently connected with each other via rigid linkers. Such covalent cucurbituril networks might be useful for the capture of radioactive Cs-137 (present as Cs{sup +}) in the contaminated environment.

Comparative Analysis of Pharmacophore Features and Quantitative Structure-Activity Relationships for CD38 Covalent and Non-covalent Inhibitors.

PubMed

Zhang, Shuang; Xue, Xiwen; Zhang, Liangren; Zhang, Lihe; Liu, Zhenming

2015-12-01

In the past decade, the discovery, synthesis, and evaluation for hundreds of CD38 covalent and non-covalent inhibitors has been reported sequentially by our group and partners; however, a systematic structure-based guidance is still lacking for rational design of CD38 inhibitor. Here, we carried out a comparative analysis of pharmacophore features and quantitative structure-activity relationships for CD38 inhibitors. The results uncover that the essential interactions between key residues and covalent/non-covalent CD38 inhibitors include (i) hydrogen bond and hydrophobic interactions with residues Glu226 and Trp125, (ii) electrostatic or hydrogen bond interaction with the positively charged residue Arg127 region, and (iii) the hydrophobic interaction with residue Trp189. For covalent inhibitors, besides the covalent effect with residue Glu226, the electrostatic interaction with residue Arg127 is also necessary, while another hydrogen/non-bonded interaction with residues Trp125 and Trp189 can also be detected. By means of the SYBYL multifit alignment function, the best CoMFA and CoMSIA with CD38 covalent inhibitors presented cross-validated correlation coefficient values (q(2)) of 0.564 and 0.571, and non-cross-validated values (r(2)) of 0.967 and 0.971, respectively. The CD38 non-covalent inhibitors can be classified into five groups according to their chemical scaffolds, and the residues Glu226, Trp189, and Trp125 are indispensable for those non-covalent inhibitors binding to CD38, while the residues Ser126, Arg127, Asp155, Thr221, and Phe222 are also important. The best CoMFA and CoMSIA with the F12 analogues presented cross-validated correlation coefficient values (q(2)) of 0.469 and 0.454, and non-cross-validated values (r(2)) of 0.814 and 0.819, respectively. © 2015 John Wiley & Sons A/S.

The histone modifications governing TFF1 transcription mediated by estrogen receptor.

PubMed

Li, Yanyan; Sun, Luyang; Zhang, Yu; Wang, Dandan; Wang, Feng; Liang, Jing; Gui, Bin; Shang, Yongfeng

2011-04-22

Transcription regulation by histone modifications is a major contributing factor to the structural and functional diversity in biology. These modifications are encrypted as histone codes or histone languages and function to establish and maintain heritable epigenetic codes that define the identity and the fate of the cell. Despite recent advances revealing numerous histone modifications associated with transcription regulation, how such modifications dictate the process of transcription is not fully understood. Here we describe spatial and temporal analyses of the histone modifications that are introduced during estrogen receptor α (ERα)-activated transcription. We demonstrated that aborting RNA polymerase II caused a disruption of the histone modifications that are associated with transcription elongation but had a minimal effect on modifications deposited during transcription initiation. We also found that the histone H3S10 phosphorylation mark is catalyzed by mitogen- and stress-activated protein kinase 1 (MSK1) and is recognized by a 14-3-3ζ/14-3-3ε heterodimer through its interaction with H3K4 trimethyltransferase SMYD3 and the p52 subunit of TFIIH. We showed that H3S10 phosphorylation is a prerequisite for H3K4 trimethylation. In addition, we demonstrated that SET8/PR-Set7/KMT5A is required for ERα-regulated transcription and its catalyzed H4K20 monomethylation is implicated in both transcription initiation and elongation. Our experiments provide a relatively comprehensive analysis of histone modifications associated with ERα-regulated transcription and define the biological meaning of several key components of the histone code that governs ERα-regulated transcription.

Characterization of the covalent binding of N-phenyl-N'-(2-chloroethyl)ureas to {beta}-tubulin: importance of Glu198 in microtubule stability.

PubMed

Fortin, Sébastien; Bouchon, Bernadette; Chambon, Christophe; Lacroix, Jacques; Moreau, Emmanuel; Chezal, Jean-Michel; Degoul, Françoise; C-Gaudreault, René

2011-02-01

N-Phenyl-N'-(2-chloroethyl)ureas (CEUs) are antimicrotubule agents interacting covalently with β-tubulin near the colchicine-binding site (C-BS). Glutamyl 198 residue in β-tubulin (Glu198), which is adjacent to the C-BS behind the two potent nucleophilic residues, Cys239 and Cys354, has been shown to covalently react with 1-(2-chloroethyl)-3-(4-iodophenyl)urea (ICEU). By use of mass spectrometry, we have now identified residues in β-tubulin that have become modified irreversibly by 1-(2-chloroethyl)-3-[3-(5-hydroxypentyl)phenyl]urea (HPCEU), 1-[4-(3-hydroxy-4-methoxystyryl)phenyl]-3-(2-chloroethyl)urea (4ZCombCEU), and N,N'-ethylenebis(iodoacetamide) (EBI). The binding of HPCEU and 4ZCombCEU to β-tubulin resulted in the acylation of Glu198, a protein modification of uncommon occurrence in living cells. Prototypical CEUs then were used as molecular probes to assess, in mouse B16F0 and human MDA-MB-231 cells, the role of Glu198 in microtubule stability. For that purpose, we studied the effect of Glu198 modification by ICEU, HPCEU, and 4ZCombCEU on the acetylation of Lys40 on α-tubulin, a key indicator of microtubule stability. We show that modification of Glu198 by prototypical CEUs correlates with a decrease in Lys40 acetylation, as observed also with other microtubule depolymerizing agents. Therefore, CEU affects the stability and the dynamics of microtubule, likewise a E198G mutation, which is unusual for xenobiotics. We demonstrate for the first time that EBI forms an intramolecular cross-link between Cys239 and Cys354 of β-tubulin in living cells. This work establishes a novel basis for the development of future chemotherapeutic agents and provides a framework for the design of molecules useful for studying the role of Asp and Glu residues in the structure/function and the biological activity of several cellular proteins under physiological conditions.

The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

PubMed Central

Cross, Janet V; Foss, Frank W; Rady, Joshua M; Macdonald, Timothy L; Templeton, Dennis J

2007-01-01

Background Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway. Methods The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases. Results ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected. Conclusion These results demonstrate that MEKK1 is directly

Germline mutations affecting the histone H4 core cause a developmental syndrome by altering DNA damage response and cell cycle control.

PubMed

Tessadori, Federico; Giltay, Jacques C; Hurst, Jane A; Massink, Maarten P; Duran, Karen; Vos, Harmjan R; van Es, Robert M; Scott, Richard H; van Gassen, Koen L I; Bakkers, Jeroen; van Haaften, Gijs

2017-11-01

Covalent modifications of histones have an established role as chromatin effectors, as they control processes such as DNA replication and transcription, and repair or regulate nucleosomal structure. Loss of modifications on histone N tails, whether due to mutations in genes belonging to histone-modifying complexes or mutations directly affecting the histone tails, causes developmental disorders or has a role in tumorigenesis. More recently, modifications affecting the globular histone core have been uncovered as being crucial for DNA repair, pluripotency and oncogenesis. Here we report monoallelic missense mutations affecting lysine 91 in the histone H4 core (H4K91) in three individuals with a syndrome of growth delay, microcephaly and intellectual disability. Expression of the histone H4 mutants in zebrafish embryos recapitulates the developmental anomalies seen in the patients. We show that the histone H4 alterations cause genomic instability, resulting in increased apoptosis and cell cycle progression anomalies during early development. Mechanistically, our findings indicate an important role for the ubiquitination of H4K91 in genomic stability during embryonic development.

ATRX tolerates activity-dependent histone H3 methyl/phos switching to maintain repetitive element silencing in neurons

PubMed Central

Noh, Kyung-Min; Zhao, Dan; Xiang, Bin; Wenderski, Wendy; Lewis, Peter W.; Shen, Li; Li, Haitao; Allis, C. David

2015-01-01

ATRX (the alpha thalassemia/mental retardation syndrome X-linked protein) is a member of the switch2/sucrose nonfermentable2 (SWI2/SNF2) family of chromatin-remodeling proteins and primarily functions at heterochromatic loci via its recognition of “repressive” histone modifications [e.g., histone H3 lysine 9 tri-methylation (H3K9me3)]. Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX’s ability to recognize an activity-dependent combinatorial histone modification, histone H3 lysine 9 tri-methylation/serine 10 phosphorylation (H3K9me3S10ph), in postmitotic neurons. In neurons, this “methyl/phos” switch occurs exclusively after periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph-bound Atrx represses noncoding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction. PMID:25538301

ATRX tolerates activity-dependent histone H3 methyl/phos switching to maintain repetitive element silencing in neurons.

PubMed

Noh, Kyung-Min; Maze, Ian; Zhao, Dan; Xiang, Bin; Wenderski, Wendy; Lewis, Peter W; Shen, Li; Li, Haitao; Allis, C David

2015-06-02

ATRX (the alpha thalassemia/mental retardation syndrome X-linked protein) is a member of the switch2/sucrose nonfermentable2 (SWI2/SNF2) family of chromatin-remodeling proteins and primarily functions at heterochromatic loci via its recognition of "repressive" histone modifications [e.g., histone H3 lysine 9 tri-methylation (H3K9me3)]. Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX's ability to recognize an activity-dependent combinatorial histone modification, histone H3 lysine 9 tri-methylation/serine 10 phosphorylation (H3K9me3S10ph), in postmitotic neurons. In neurons, this "methyl/phos" switch occurs exclusively after periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph-bound Atrx represses noncoding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction.

Covalent inhibitors: an opportunity for rational target selectivity.

PubMed

Lagoutte, Roman; Patouret, Remi; Winssinger, Nicolas

2017-08-01

There is a resurging interest in compounds that engage their target through covalent interactions. Cysteine's thiol is endowed with enhanced reactivity, making it the nucleophile of choice for covalent engagement with a ligand aligning an electrophilic trap with a cysteine residue in a target of interest. The paucity of cysteine in the proteome coupled to the fact that closely related proteins do not necessarily share a given cysteine residue enable a level of unprecedented rational target selectivity. The recent demonstration that a lysine's amine can also be engaged covalently with a mild electrophile extends the potential of covalent inhibitors. The growing database of protein structures facilitates the discovery of covalent inhibitors while the advent of proteomic technologies enables a finer resolution in the selectivity of covalently engaged proteins. Here, we discuss recent examples of discovery and design of covalent inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Covalent bonding in heavy metal oxides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bagus, Paul S.; Nelin, Connie J.; Hrovat, Dave A.

Novel theoretical methods were used to quantify the magnitude and the energetic contributions of 4f/5f-O2p and 5d/6d-O2p interactions to covalent bonding in lanthanide and actinide oxides. Although many analyses have neglected the involvement of the frontier d orbitals, the present study shows that f and d covalency are of comparable importance. Two trends are identified. As is expected, the covalent mixing is larger when the nominal oxidation state is higher. More subtly, the importance of the nf covalent mixing decreases sharply relative to (n+1)d as the nf occupation increases. Atomic properties of the metal cations that drive these trends aremore » identified.« less

Highly stable ionic-covalent cross-linked sulfonated poly(ether ether ketone) for direct methanol fuel cells

NASA Astrophysics Data System (ADS)

Lei, Linfeng; Zhu, Xingye; Xu, Jianfeng; Qian, Huidong; Zou, Zhiqing; Yang, Hui

2017-05-01

A novel ionic cross-linked sulfonated poly(ether ether ketone) containing equal content of sulfonic acid and pendant tertiary amine groups (TA-SPEEK) has been initially synthesized for the application in direct methanol fuel cells (DMFCs). By adjusting the ratio of p-xylene dibromide to tertiary amine groups of TA-SPEEK, a series of ionic-covalent cross-linked membranes (C-SPEEK-x) with tunable degree of cross-linking are prepared. Compared with the pristine membrane, the ionic and ionic-covalent cross-linked proton exchange membranes (PEMs) exhibit reduced methanol permeability and improved mechanical properties, dimensional and oxidative stability. The proton conductivity and methanol selectivity of protonated TA-SPEEK and C-SPEEK-x at 25 °C is up to 0.109 S cm-1 and 3.88 × 105 S s cm-3, respectively, which are higher than that of Nafion 115. The DMFC incorporating C-SPEEK-25 exhibits a maximum power density as high as 35.3 mW cm-2 with 4 M MeOH at 25 °C (31.8 mW cm-2 for Nafion 115). Due to the highly oxidative stability of the membrane, no obvious performance degradation of the DMFC is observed after more than 400 h operation, indicating such cost-effective ionic-covalent cross-linked membranes have substantial potential as alternative PEMs for DMFC applications.

Tough Self-Healing Elastomers by Molecular Enforced Integration of Covalent and Reversible Networks.

PubMed

Wu, Jinrong; Cai, Li-Heng; Weitz, David A

2017-10-01

Self-healing polymers crosslinked by solely reversible bonds are intrinsically weaker than common covalently crosslinked networks. Introducing covalent crosslinks into a reversible network would improve mechanical strength. It is challenging, however, to apply this concept to "dry" elastomers, largely because reversible crosslinks such as hydrogen bonds are often polar motifs, whereas covalent crosslinks are nonpolar motifs. These two types of bonds are intrinsically immiscible without cosolvents. Here, we design and fabricate a hybrid polymer network by crosslinking randomly branched polymers carrying motifs that can form both reversible hydrogen bonds and permanent covalent crosslinks. The randomly branched polymer links such two types of bonds and forces them to mix on the molecular level without cosolvents. This enables a hybrid "dry" elastomer that is very tough with fracture energy 13500 Jm -2 comparable to that of natural rubber. Moreover, the elastomer can self-heal at room temperature with a recovered tensile strength 4 MPa, which is 30% of its original value, yet comparable to the pristine strength of existing self-healing polymers. The concept of forcing covalent and reversible bonds to mix at molecular scale to create a homogenous network is quite general and should enable development of tough, self-healing polymers of practical usage. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Caenorhabditis elegans as a model system to study post-translational modifications of human transthyretin

NASA Astrophysics Data System (ADS)

Henze, Andrea; Homann, Thomas; Rohn, Isabelle; Aschner, Michael; Link, Christopher D.; Kleuser, Burkhard; Schweigert, Florian J.; Schwerdtle, Tanja; Bornhorst, Julia

2016-11-01

The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.

Unifying Principles of the Reductive Covalent Graphene Functionalization.

PubMed

Abellán, Gonzalo; Schirowski, Milan; Edelthalhammer, Konstantin F; Fickert, Michael; Werbach, Katharina; Peterlik, Herwig; Hauke, Frank; Hirsch, Andreas

2017-04-12

Covalently functionalized graphene derivatives were synthesized via benchmark reductive routes using graphite intercalation compounds (GICs), in particular KC 8 . We have compared the graphene arylation and alkylation of the GIC using 4-tert-butylphenyldiazonium and bis(4-(tert-butyl)phenyl)iodonium salts, as well as phenyl iodide, n-hexyl iodide, and n-dodecyl iodide, as electrophiles in model reactions. We have put a particular focus on the evaluation of the degree of addition and the bulk functionalization homogeneity (H bulk ). For this purpose, we have employed statistical Raman spectroscopy (SRS), and a forefront characterization tool using thermogravimetric analysis coupled with FT-IR, gas chromatography, and mass spectrometry (TGA/FT-IR/GC/MS). The present study unambiguously shows that the graphene functionalization using alkyl iodides leads to the best results, in terms of both the degree of addition and the H bulk . Moreover, we have identified the reversible character of the covalent addition chemistry, even at temperatures below 200 °C. The thermally induced addend cleavage proceeds homolytically, which allows for the detection of dimeric cleavage products by TGA/FT-IR/GC/MS. This dimerization points to a certain degree of regioselectivity, leading to a low sheet homogeneity (H sheet ). Finally, we developed this concept by performing the reductive alkylation reaction in monolayer CVD graphene films. This work provides important insights into the understanding of basic principles of reductive graphene functionalization and will serve as a guide in the design of new graphene functionalization concepts.

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*

PubMed Central

Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.

2015-01-01

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

DOE PAGES

Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; ...

2014-12-31

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

A Simple Method for Visualization of Locus-Specific H4K20me1 Modifications in Living Caenorhabditis elegans Single Cells.

PubMed

Shinkai, Yoichi; Kuramochi, Masahiro; Doi, Motomichi

2018-05-03

Recently, advances in next-generation sequencing technologies have enabled genome-wide analyses of epigenetic modifications; however, it remains difficult to analyze the states of histone modifications at a single-cell resolution in living multicellular organisms because of the heterogeneity within cellular populations. Here we describe a simple method to visualize histone modifications on the specific sequence of target locus at a single-cell resolution in living Caenorhabditis elegans , by combining the LacO/LacI system and a genetically-encoded H4K20me1-specific probe, "mintbody". We demonstrate that Venus-labeled mintbody and mTurquoise2-labeled LacI can co-localize on an artificial chromosome carrying both the target locus and LacO sequences, where H4K20me1 marks the target locus. We demonstrate that our visualization method can precisely detect H4K20me1 depositions on the her-1 gene sequences on the artificial chromosome, to which the dosage compensation complex binds to regulate sex determination. The degree of H4K20me1 deposition on the her-1 sequences on the artificial chromosome correlated strongly with sex, suggesting that, using the artificial chromosome, this method can reflect context-dependent changes of H4K20me1 on endogenous genomes. Furthermore, we demonstrate live imaging of H4K20me1 depositions on the artificial chromosome. Combined with ChIP assays, this mintbody-LacO/LacI visualization method will enable analysis of developmental and context-dependent alterations of locus-specific histone modifications in specific cells and elucidation of the underlying molecular mechanisms. Copyright © 2018, G3: Genes, Genomes, Genetics.

Green electrochemical modification of RVC foam electrode and improved H2O2 electrogeneration by applying pulsed current for pollutant removal.

PubMed

Zhou, Wei; Ding, Yani; Gao, Jihui; Kou, Kaikai; Wang, Yan; Meng, Xiaoxiao; Wu, Shaohua; Qin, Yukun

2018-02-01

The performance of cathode on H 2 O 2 electrogeneration is a critical factor that limits the practical application of electro-Fenton (EF) process. Herein, we report a simple but effective electrochemical modification of reticulated vitreous carbon foam (RVC foam) electrode for enhanced H 2 O 2 electrogeneration. Cyclic voltammetry, chronoamperometry, and X-ray photoelectron spectrum were used to characterize the modified electrode. Oxygen-containing groups (72.5-184.0 μmol/g) were introduced to RVC foam surface, thus resulting in a 59.8-258.2% higher H 2 O 2 yield. The modified electrodes showed much higher electrocatalytic activity toward O 2 reduction and good stability. Moreover, aimed at weakening the extent of electroreduction of H 2 O 2 in porous RVC foam, the strategy of pulsed current was proposed. H 2 O 2 concentration was 582.3 and 114.0% higher than the unmodified and modified electrodes, respectively. To test the feasibility of modification, as well as pulsed current, EF process was operated for removal of Reactive Blue 19 (RB19). The fluorescence intensity of hydroxybenzoic acid in EF with modified electrode is 3.2 times higher than EF with unmodified electrode, illustrating more hydroxyl radicals were generated. The removal efficiency of RB 19 in EF with unmodified electrode, modified electrode, and unmodified electrode assisted by pulsed current was 53.9, 68.9, and 81.1%, respectively, demonstrating that the green modification approach, as well as pulsed current, is applicable in EF system for pollutant removal. Graphical abstract ᅟ.

Disintegration-controllable stimuli-responsive polyelectrolyte multilayer microcapsules via covalent layer-by-layer assembly.

PubMed

Mu, Bin; Lu, Chunyin; Liu, Peng

2011-02-01

The disintegration-controllable stimuli-responsive polyelectrolyte multilayer microcapsules have been fabricated via the covalent layer-by-layer assembly between the amino groups of chitosan (CS) and the aldehyde groups of the oxidized sodium alginate (OSA) onto the sacrificial templates (polystyrene sulfonate, PSS) which was removed by dialysis subsequently. The covalent crosslinking bonds of the multilayer microcapsules were confirmed by FTIR analysis. The TEM analysis showed that the diameter of the multilayer microcapsules was <200nm. The diameter of the multilayer microcapsules decreased with the increasing of the pH values or the ionic strength. The pH and ionic strength dual-responsive multilayer microcapsules were stable in acidic and neutral media while they could disintegrate only at strong basic media. Copyright © 2010 Elsevier B.V. All rights reserved.

Investigating the Non-Covalent Functionalization and Chemical Transformation of Graphene

NASA Astrophysics Data System (ADS)

Sham, Chun-Hong

Trend in device miniatures demands capabilities to produce rationally designed patterns in ever-shrinking length scale. The research community has examined various techniques to push the current lithography resolution to sub-10nm scale. One of the ideas is to utilize the natural nanoscale patterns of molecular assemblies. In this thesis, the self-assembling phenomenon of a photoactive molecule on epitaxial graphene (EG) grown on SiC was discussed. This molecular assembly enables manipulation of chemical contrast in nanoscale through UV exposure or atomic layer deposition. Future development of nanoelectronics industry will be fueled by innovations in electronics materials, which could be discovered through covalent modification of graphene. In a study reported in this thesis, silicon is deposited onto EG. After annealing, a new surface reconstruction, identified to be (3x3)-SiC, was formed. Raman spectroscopy finds no signature of graphene after annealing, indicating a complete chemical transformation of graphene. DFT calculations reveal a possible conversion mechanism. Overall, these studies provide insights for future device miniaturization; contribute to the search of novel materials and help bridging the gap between graphene and current silicon-based industrial infrastructures.

Natural variation of H3K27me3 distribution between two Arabidopsis accessions and its association with flanking transposable elements

PubMed Central

2012-01-01

Background Histone H3 lysine 27 tri-methylation and lysine 9 di-methylation are independent repressive chromatin modifications in Arabidopsis thaliana. H3K27me3 is established and maintained by Polycomb repressive complexes whereas H3K9me2 is catalyzed by SUVH histone methyltransferases. Both modifications can spread to flanking regions after initialization and were shown to be mutually exclusive in Arabidopsis. Results We analyzed the extent of natural variation of H3K27me3 in the two accessions Landsberg erecta (Ler) and Columbia (Col) and their F1 hybrids. The majority of H3K27me3 target genes in Col were unchanged in Ler and F1 hybrids. A small number of Ler-specific targets were detected and confirmed. Consistent with a cis-regulatory mechanism for establishing H3K27me3, differential targets showed allele-specific H3K27me3 in hybrids. Five Ler-specific targets showed the active mark H3K4me3 in Col and for this group, differential H3K27me3 enrichment accorded to expression variation. On the other hand, the majority of Ler-specific targets were not expressed in Col, Ler or 17 other accessions. Instead of H3K27me3, the antagonistic mark H3K9me2 and other heterochromatic features were observed at these loci in Col. These loci were frequently flanked by transposable elements, which were often missing in the Ler genome assembly. Conclusion There is little variation in H3K27me3 occupancy within the species, although H3K27me3 targets were previously shown as overrepresented among differentially expressed genes. The existing variation in H3K27me3 seems mostly explained by flanking polymorphic transposable elements. These could nucleate heterochromatin, which then spreads into neighboring H3K27me3 genes, thus converting them to H3K9me2 targets. PMID:23253144

Inactivation of prion proteins via covalent grafting with methoxypoly(ethylene glycol).

PubMed

Scott, Mark D

2006-01-01

Transmissible spongiform encephalopathies (TSE) such as bovine spongiform encephalitis (BSE), Creutzfeld-Jakob disease (CJD) as well as other proteinaceous infectious particles (prions) mediated diseases have emerged as a significant concern in transfusion medicine. This concern is derived from both the disease causing potential of prion contaminated blood products but also due to tremendous impact of the active deferral of current and potential blood donors due to their extended stays in BSE prevalent countries (e.g., the United Kingdom). To date, there are no effective means by which infectious prion proteins can be inactivated in cellular and acellular blood products. Based on current work on the covalent grafting of methoxypoly(ethylene glycol) [mPEG] to proteins, viruses, and anuclear, and nucleated cells, it is hypothesized that the conversion of the normal PrP protein to its mutant conformation can be prevented by the covalent grafting of mPEG to the mutant protein. Inactivation of infective protein particles (prions) in both cellular blood products as well as cell free solutions (e.g., clotting factors) could be of medical/commercial value. It is hypothesized that consequent to the covalent modification of donor-derived prions with mPEG the requisite nucleation of the normal and mutant PrP proteins is inhibited due to the increased solubility of the modified mutant PrP and that the conformational conversion arising from the mutant PrP is prevented due to obscuration of protein charge by the heavily hydrated and neutral mPEG polymers, as well as by direct steric hindrance of the interaction due to the highly mobile polymer graft.

Protein Arginine Methyltransferase 7 Regulates Cellular Response to DNA Damage by Methylating Promoter Histones H2A and H4 of the Polymerase δ Catalytic Subunit Gene, POLD1*

PubMed Central

Karkhanis, Vrajesh; Wang, Li; Tae, Sookil; Hu, Yu-Jie; Imbalzano, Anthony N.; Sif, Saïd

2012-01-01

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage. PMID:22761421

Protein arginine methyltransferase 7 regulates cellular response to DNA damage by methylating promoter histones H2A and H4 of the polymerase δ catalytic subunit gene, POLD1.

PubMed

Karkhanis, Vrajesh; Wang, Li; Tae, Sookil; Hu, Yu-Jie; Imbalzano, Anthony N; Sif, Saïd

2012-08-24

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.

Covalently Linked Tandem Lesions in DNA

PubMed Central

Patrzyc, Helen B.; Dawidzik, Jean B.; Budzinski, Edwin E.; Freund, Harold G.; Wilton, John H.; Box, Harold C.

2013-01-01

Reactive oxygen species (ROS) generate a type of DNA damage called tandem lesions, two adjacent nucleotides both modified. A subcategory of tandem lesions consists of adjacent nucleotides linked by a covalent bond. Covalently linked tandem lesions generate highly characteristic liquid chromotography-tandem mass spectrometry (LC-MS/MS) elution profiles. We have used this property to comprehensively survey X-irradiated DNA for covalently linked tandem lesions. A total of 15 tandem lesions were detected in DNA irradiated in deoxygenated aqueous solution, five tandem lesions were detected in DNA that was irradiated in oxygenated solution. PMID:23106212

Mixed non-covalent assemblies of ethynyl nile red and ethynyl pyrene along oligonucleotide templates.

PubMed

Ensslen, Philipp; Fritz, Yannic; Wagenknecht, Hans-Achim

2015-01-14

Ethynyl pyrene and ethynyl nile red as modifications at the 5-position of 2'-deoxyuridines self-assemble non-covalently and specifically along oligo-2'-deoxyadenosines as templates. Oligo-2'-deoxyadenosines of the lengths (dA)10-(dA)20 are able to retain nearly exactly as many ethynyl nile red units in solution as binding sites are available on these templates. In contrast, in the presence of oligo-2'-thymidines the ethynyl nile red moieties are similarly insoluble to those in the absence of any oligonucleotide and yield an aggregate. The mixed assemblies of both chromophores are highly ordered, show left-handed chirality and yield dual fluorescence. The strong excitonic coupling indicates assemblies with a high degree of order. These results show that DNA represents an important supramolecular scaffold for the templated, helical and non-covalent arrangement not only for one type of chromophore but also for mixtures of two different chromophores.

Hydrogels with covalent and noncovalent crosslinks

NASA Technical Reports Server (NTRS)

Kilck, Kristi L. (Inventor); Yamaguchi, Nori (Inventor)

2013-01-01

A method for targeted delivery of therapeutic compounds from hydrogels is presented. The method involves administering to a cell a hydrogel in which a therapeutic compound is noncovalently bound to heparin. The hydrogel may contain covalent and non-covalent crosslinks.

Improved modification for the density-functional theory calculation of thermodynamic properties for C-H-O composite compounds.

PubMed

Liu, Min Hsien; Chen, Cheng; Hong, Yaw Shun

2005-02-08

A three-parametric modification equation and the least-squares approach are adopted to calibrating hybrid density-functional theory energies of C(1)-C(10) straight-chain aldehydes, alcohols, and alkoxides to accurate enthalpies of formation DeltaH(f) and Gibbs free energies of formation DeltaG(f), respectively. All calculated energies of the C-H-O composite compounds were obtained based on B3LYP6-311++G(3df,2pd) single-point energies and the related thermal corrections of B3LYP6-31G(d,p) optimized geometries. This investigation revealed that all compounds had 0.05% average absolute relative error (ARE) for the atomization energies, with mean value of absolute error (MAE) of just 2.1 kJ/mol (0.5 kcal/mol) for the DeltaH(f) and 2.4 kJ/mol (0.6 kcal/mol) for the DeltaG(f) of formation.

Effective scheme for partitioning covalent bonds in density-functional embedding theory: From molecules to extended covalent systems.

PubMed

Huang, Chen; Muñoz-García, Ana Belén; Pavone, Michele

2016-12-28

Density-functional embedding theory provides a general way to perform multi-physics quantum mechanics simulations of large-scale materials by dividing the total system's electron density into a cluster's density and its environment's density. It is then possible to compute the accurate local electronic structures and energetics of the embedded cluster with high-level methods, meanwhile retaining a low-level description of the environment. The prerequisite step in the density-functional embedding theory is the cluster definition. In covalent systems, cutting across the covalent bonds that connect the cluster and its environment leads to dangling bonds (unpaired electrons). These represent a major obstacle for the application of density-functional embedding theory to study extended covalent systems. In this work, we developed a simple scheme to define the cluster in covalent systems. Instead of cutting covalent bonds, we directly split the boundary atoms for maintaining the valency of the cluster. With this new covalent embedding scheme, we compute the dehydrogenation energies of several different molecules, as well as the binding energy of a cobalt atom on graphene. Well localized cluster densities are observed, which can facilitate the use of localized basis sets in high-level calculations. The results are found to converge faster with the embedding method than the other multi-physics approach ONIOM. This work paves the way to perform the density-functional embedding simulations of heterogeneous systems in which different types of chemical bonds are present.

The anticancer natural product ophiobolin A induces cytotoxicity by covalent modification of phosphatidylethanolamine.

PubMed

Chidley, Christopher; Trauger, Sunia A; Birsoy, Kıvanç; O'Shea, Erin K

2016-07-12

Phenotypic screens allow the identification of small molecules with promising anticancer activity, but the difficulty in characterizing the mechanism of action of these compounds in human cells often undermines their value as drug leads. Here, we used a loss-of-function genetic screen in human haploid KBM7 cells to discover the mechanism of action of the anticancer natural product ophiobolin A (OPA). We found that genetic inactivation of de novo synthesis of phosphatidylethanolamine (PE) mitigates OPA cytotoxicity by reducing cellular PE levels. OPA reacts with the ethanolamine head group of PE in human cells to form pyrrole-containing covalent cytotoxic adducts and these adducts lead to lipid bilayer destabilization. Our characterization of this unusual cytotoxicity mechanism, made possible by unbiased genetic screening in human cells, suggests that the selective antitumor activity displayed by OPA may be due to altered membrane PE levels in cancer cells.

The Impact of Sonication on the Surface Quality of Single-Walled Carbon Nanotubes.

PubMed

Koh, Byumseok; Cheng, Wei

2015-08-01

Sonication process is regularly adopted for dispersing single-walled carbon nanotubes (SWCNTs) in an aqueous medium. This can be achieved by either covalent functionalization of SWCNTs with strong acid or by noncovalent functionalization using dispersants that adsorb onto the surface of SWCNTs during dispersion. Because the dispersion process is usually performed using sonication, unintentional free radical formation during sonication process may induce covalent modification of SWCNT surface. Herein, we have systematically investigated the status of SWCNT surface modification under various sonication conditions using Raman spectroscopy. Comparing ID /IG (Raman intensities between D and G bands) ratio of SWCNTs under various sonication conditions suggests that typical sonication conditions (1-6 h bath sonication with sonication power between 3 and 80 W) in aqueous media do not induce covalent modification of SWCNT surface. In addition, we confirm that SWCNT dispersion with single-stranded DNA (ssDNA) involves noncovalent adsorption of ssDNA onto the surface of SWCNTs, but not covalent linkage between ssDNA and SWCNT surface. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Phosphorylation of histone H3(T118) alters nucleosome dynamics and remodeling

PubMed Central

North, Justin A.; Javaid, Sarah; Ferdinand, Michelle B.; Chatterjee, Nilanjana; Picking, Jonathan W.; Shoffner, Matthew; Nakkula, Robin J.; Bartholomew, Blaine; Ottesen, Jennifer J.; Fishel, Richard; Poirier, Michael G.

2011-01-01

Nucleosomes, the fundamental units of chromatin structure, are regulators and barriers to transcription, replication and repair. Post-translational modifications (PTMs) of the histone proteins within nucleosomes regulate these DNA processes. Histone H3(T118) is a site of phosphorylation [H3(T118ph)] and is implicated in regulation of transcription and DNA repair. We prepared H3(T118ph) by expressed protein ligation and determined its influence on nucleosome dynamics. We find H3(T118ph) reduces DNA–histone binding by 2 kcal/mol, increases nucleosome mobility by 28-fold and increases DNA accessibility near the dyad region by 6-fold. Moreover, H3(T118ph) increases the rate of hMSH2–hMSH6 nucleosome disassembly and enables nucleosome disassembly by the SWI/SNF chromatin remodeler. These studies suggest that H3(T118ph) directly enhances and may reprogram chromatin remodeling reactions. PMID:21576235

SIRT3 restricts HBV transcription and replication via epigenetic regulation of cccDNA involving SUV39H1 and SETD1A histone methyltransferases.

PubMed

Ren, Ji-Hua; Hu, Jie-Li; Cheng, Sheng-Tao; Yu, Hai-Bo; Wong, Vincent Kam Wai; Law, Betty Yuen Kwan; Yang, Yong-Feng; Huang, Ying; Liu, Yi; Chen, Wei-Xian; Cai, Xue-Fei; Tang, Hua; Hu, Yuan; Zhang, Wen-Lu; Liu, Xiang; Long, Quan-Xin; Zhou, Li; Tao, Na-Na; Zhou, Hong-Zhong; Yang, Qiu-Xia; Ren, Fang; He, Lin; Gong, Rui; Huang, Ai-Long; Chen, Juan

2018-04-06

Hepatitis B virus (HBV) infection remains a major health problem worldwide. Maintenance of the covalently closed circular DNA (cccDNA) which serves as a template for HBV RNA transcription is responsible for the failure of eradicating chronic HBV during current antiviral therapy. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications. In this study, we identified SIRT3 as a host factor restricting HBV transcription and replication by screening seven members of Sirtuin family which is the class III histone deacetylase. Ectopic SIRT3 expression significantly reduced total HBV RNAs, 3.5-kb RNA as well as replicative intermediate DNA in HBV-infected HepG2-NTCP cells and PHH. In contrast, gene silencing of SIRT3 promoted HBV transcription and replication. Mechanistic study found nuclear SIRT3 was recruited to the HBV cccDNA, where it deacetylated histone 3 lysine 9 (H3K9). Importantly, occupancy of SIRT3 onto cccDNA could increase the recruitment of histone methyltransferase SUV39H1 to cccDNA and decrease recruitment of SETD1A, leading to a marked increase of H3K9me3 and a decrease of H3K4me3 on cccDNA. Moreover, SIRT3-mediated HBV cccDNA transcriptional repression involved decreased binding of host RNA polymerase II and transcription factor YY1 to cccDNA. Finally, viral protein HBx could relieve SIRT3-mediated cccDNA transcriptional repression by inhibiting both SIRT3 expression and its recruitment to cccDNA. SIRT3 is a novel host factor epigenetically restricting HBV cccDNA transcription by acting cooperatively with histone methyltransferase. These data provided a rational for the use of SIRT3 activators in the prevention or treatment of HBV infection. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

Discovery of host-targeted covalent inhibitors of dengue virus

PubMed Central

de Wispelaere, Mélissanne; Carocci, Margot; Liang, Yanke; Liu, Qingsong; Sun, Eileen; Vetter, Michael L.; Wang, Jinhua; Gray, Nathanael S.; Yang, Priscilla L.

2017-01-01

We report here on an approach targeting the host reactive cysteinome to identify inhibitors of host factors required for the infectious cycle of Flaviviruses and other viruses. We used two parallel cellular phenotypic screens to identify a series of covalent inhibitors, exemplified by QL-XII-47, that are active against dengue virus. We show that the compounds effectively block viral protein expression and that this inhibition is associated with repression of downstream processes of the infectious cycle, and thus significantly contributes to the potent antiviral activity of these compounds. We demonstrate that QL-XII-47’s antiviral activity requires selective, covalent modification of a host target by showing that the compound's antiviral activity is recapitulated when cells are preincubated with QL-XII-47 and then washed prior to viral infection and by showing that QL-XII-47R, a non-reactive analog, lacks antiviral activity at concentrations more than 20-fold higher than QL-XII-47's IC90. QL-XII-47’s inhibition of Zika virus, West Nile virus, hepatitis C virus, and poliovirus further suggests that it acts via a target mediating inhibition of these other medically relevant viruses. These results demonstrate the utility of screens targeting the host reactive cysteinome for rapid identification of compounds with potent antiviral activity. PMID:28034743

Photo-reduction of CO2 Using a Rhenium Complex Covalently Supported on a Graphene/TiO2 Composite.

PubMed

Cui, Shi-Cong; Sun, Xue-Zhong; Liu, Jin-Gang

2016-07-07

One of the promising solutions for decreasing atmospheric CO2 is artificial photosynthesis, in which CO2 can be photoconverted into solar fuels. In this study, a rhenium complex Re(PyBn)(CO)3 Cl (PyBn=1-(2-picolyl)-4-phenyl-1H-1,2,3-triazole) was covalently grafted onto the surface of reduced graphene oxide (rGO). This was further combined with TiO2 to fabricate a novel catalyst composite TiO2 -rGO-Re(PyBn)(CO)3 Cl for CO2 photo-reduction. This hybrid composite demonstrated high selectivity conversion of CO2 into CO under xenon-lamp irradiation. Compared with the unsupported homogeneous catalyst Re(PyBn)(CO)3 Cl, the covalent immobilized catalyst composite TiO2 -rGO-Re(PyBn)(CO)3 Cl enhanced the turnover number six times and significantly improved catalyst stability. During the process of CO2 photo-reduction, intermediate species with lifetimes longer than hundreds of microseconds were observed and the formation of CO products was revealed using timeresolved infrared spectroscopy. A plausible mechanism for CO2 photo-reduction by the TiO2 -rGO-Re(PyBn)(CO)3 Cl catalyst composite has been suggested. The obtained results have implications for the future design of efficient catalyst composites for CO2 photo-conversion. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Low damage electrical modification of 4H-SiC via ultrafast laser irradiation

NASA Astrophysics Data System (ADS)

Ahn, Minhyung; Cahyadi, Rico; Wendorf, Joseph; Bowen, Willie; Torralva, Ben; Yalisove, Steven; Phillips, Jamie

2018-04-01

The electrical properties of 4H-SiC under ultrafast laser irradiation in the low fluence regime (<0.50 J/cm2) are presented. The appearance of high spatial frequency laser induced periodic surface structures is observed at a fluence near 0.25 J/cm2 and above, with variability in environments like in air, nitrogen, and a vacuum. In addition to the formation of periodic surface structures, ultrafast laser irradiation results in possible surface oxidation and amorphization of the material. Lateral conductance exhibits orders of magnitude increase, which is attributed to either surface conduction or modification of electrical contact properties, depending on the initial material conductivity. Schottky barrier formation on ultrafast laser irradiated 4H-SiC shows an increase in the barrier height, an increase in the ideality factor, and sub-bandgap photovoltaic responses, suggesting the formation of photo-active point defects. The results suggest that the ultrafast laser irradiation technique provides a means of engineering spatially localized structural and electronic modification of wide bandgap materials such as 4H-SiC with relatively low surface damage via low temperature processing.

Separation of Arylenevinylene Macrocycles with a Surface-Confined Two-Dimensional Covalent Organic Framework.

PubMed

Liu, Chunhua; Park, Eunsol; Jin, Yinghua; Liu, Jie; Yu, Yanxia; Zhang, Wei; Lei, Shengbin; Hu, Wenping

2018-05-31

A two-dimensional surface covalent organic framework, prepared by a surface-confined synthesis using 4,4'-azodianiline and benzene-1,3,5-tricarbaldehyde as the precursors, was used as a host network to effectively immobilize arylenevinylene macrocycles (AVMs). Thus AVMs could be separated from their linear polymer analogues, which are the common side-products in the cyclooligomerization process. Scanning tunneling microscopy investigations revealed efficient removal of linear polymers by a simple surface binding and solvent washing process. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemistry of Covalent Organic Frameworks.

PubMed

Waller, Peter J; Gándara, Felipe; Yaghi, Omar M

2015-12-15

Linking organic molecules by covalent bonds into extended solids typically generates amorphous, disordered materials. The ability to develop strategies for obtaining crystals of such solids is of interest because it opens the way for precise control of the geometry and functionality of the extended structure, and the stereochemical orientation of its constituents. Covalent organic frameworks (COFs) are a new class of porous covalent organic structures whose backbone is composed entirely of light elements (B, C, N, O, Si) that represent a successful demonstration of how crystalline materials of covalent solids can be achieved. COFs are made by combination of organic building units covalently linked into extended structures to make crystalline materials. The attainment of crystals is done by several techniques in which a balance is struck between the thermodynamic reversibility of the linking reactions and their kinetics. This success has led to the expansion of COF materials to include organic units linked by these strong covalent bonds: B-O, C-N, B-N, and B-O-Si. Since the organic constituents of COFs, when linked, do not undergo significant change in their overall geometry, it has been possible to predict the structures of the resulting COFs, and this advantage has facilitated their characterization using powder X-ray diffraction (PXRD) techniques. It has also allowed for the synthesis of COF structures by design and for their formation with the desired composition, pore size, and aperture. In practice, the modeled PXRD pattern for a given expected COF is compared with the experimental one, and depending on the quality of the match, this is used as a starting point for solving and then refining the crystal structure of the target COF. These characteristics make COFs an attractive class of new porous materials. Accordingly, they have been used as gas storage materials for energy applications, solid supports for catalysis, and optoelectronic devices. A large and

Nondestructive covalent functionalization of carbon nanotubes by selective oxidation of the original defects with K2FeO4

NASA Astrophysics Data System (ADS)

Zhang, Zhao-yang; Xu, Xue-cheng

2015-08-01

Chemical oxidation is still the major approach to the covalent functionalization of carbon nanotubes (CNTs). Theoretically, the defects on CNTs are more reactive than skeletal hexagons and should be preferentially oxidized, but conventional oxidation methods, e.g., HNO3/H2SO4 treatment, have poor reaction selectivity and inevitably consume the Cdbnd C bonds in the hexagonal lattices, leading to structural damage, π-electrons loss and weight decrease. In this work, we realized the nondestructive covalent functionalization of CNTs by selective oxidation of the defects. In our method, potassium ferrate K2FeVIO4 was employed as an oxidant for CNTs in H2SO4 medium. The CNT samples, before and after K2FeO4/H2SO4 treatment, were characterized with colloid dispersibility, IR, Raman spectroscopy, FESEM and XPS. The results indicated that (i) CNTs could be effectively oxidized by Fe (VI) under mild condition (60 °C, 3 h), and hydrophilic CNTs with abundant surface sbnd COOH groups were produced; and (ii) Fe (VI) oxidation of CNTs followed a defect-specific oxidation process, that is, only the sp3-hybridized carbon atoms on CNT surface were oxidized while the Cdbnd C bonds remained unaffected. This selective/nondestructive oxidation afforded oxidized CNTs in yields of above 100 wt%. This paper shows that K2FeO4/H2SO4 is an effective, nondestructive and green oxidation system for oxidative functionalization of CNTs and probably other carbon materials as well.

Exploring Covalent Allosteric Inhibition of Antigen 85C from Mycobacterium tuberculosis by Ebselen Derivatives

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goins, Christopher M.; Dajnowicz, Steven; Thanna, Sandeep

Previous studies identified ebselen as a potent in vitro and in vivo inhibitor of the Mycobacterium tuberculosis ( Mtb) antigen 85 (Ag85) complex, comprising three homologous enzymes required for the biosynthesis of the mycobacterial cell wall. In this study, the Mtb Ag85C enzyme was cocrystallized with azido and adamantyl ebselen derivatives, resulting in two crystallographic structures of 2.01 and 1.30 Å resolution, respectively. Both structures displayed the anticipated covalent modification of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide bond. Continuous difference density for both thiol modifiers allowed for the assessment of interactions that influence ebselen binding and inhibitormore » orientation that were unobserved in previous Ag85C ebselen structures. The k inact/ K I values for ebselen, adamantyl ebselen, and azido ebselen support the importance of observed constructive chemical interactions with Arg239 for increased in vitro efficacy toward Ag85C. To better understand the in vitro kinetic properties of these ebselen derivatives, the energetics of specific protein–inhibitor interactions and relative reaction free energies were calculated for ebselen and both derivatives using density functional theory. These studies further support the different in vitro properties of ebselen and two select ebselen derivatives from our previously published ebselen library with respect to kinetics and protein–inhibitor interactions. In both structures, the α9 helix was displaced farther from the enzyme active site than the previous Ag85C ebselen structure, resulting in the restructuring of a connecting loop and imparting a conformational change to residues believed to play a role in substrate binding specific to Ag85C. These notable structural changes directly affect protein stability, reducing the overall melting temperature by up to 14.5 °C, resulting in the unfolding of protein at physiological temperatures. Additionally

Exploring Covalent Allosteric Inhibition of Antigen 85C from Mycobacterium tuberculosis by Ebselen Derivatives

DOE PAGES

Goins, Christopher M.; Dajnowicz, Steven; Thanna, Sandeep; ...

2017-03-13

Previous studies identified ebselen as a potent in vitro and in vivo inhibitor of the Mycobacterium tuberculosis ( Mtb) antigen 85 (Ag85) complex, comprising three homologous enzymes required for the biosynthesis of the mycobacterial cell wall. In this study, the Mtb Ag85C enzyme was cocrystallized with azido and adamantyl ebselen derivatives, resulting in two crystallographic structures of 2.01 and 1.30 Å resolution, respectively. Both structures displayed the anticipated covalent modification of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide bond. Continuous difference density for both thiol modifiers allowed for the assessment of interactions that influence ebselen binding and inhibitormore » orientation that were unobserved in previous Ag85C ebselen structures. The k inact/ K I values for ebselen, adamantyl ebselen, and azido ebselen support the importance of observed constructive chemical interactions with Arg239 for increased in vitro efficacy toward Ag85C. To better understand the in vitro kinetic properties of these ebselen derivatives, the energetics of specific protein–inhibitor interactions and relative reaction free energies were calculated for ebselen and both derivatives using density functional theory. These studies further support the different in vitro properties of ebselen and two select ebselen derivatives from our previously published ebselen library with respect to kinetics and protein–inhibitor interactions. In both structures, the α9 helix was displaced farther from the enzyme active site than the previous Ag85C ebselen structure, resulting in the restructuring of a connecting loop and imparting a conformational change to residues believed to play a role in substrate binding specific to Ag85C. These notable structural changes directly affect protein stability, reducing the overall melting temperature by up to 14.5 °C, resulting in the unfolding of protein at physiological temperatures. Additionally

Exploring Covalent Allosteric Inhibition of Antigen 85C from Mycobacterium tuberculosis by Ebselen Derivatives.

PubMed

Goins, Christopher M; Dajnowicz, Steven; Thanna, Sandeep; Sucheck, Steven J; Parks, Jerry M; Ronning, Donald R

2017-05-12

Previous studies identified ebselen as a potent in vitro and in vivo inhibitor of the Mycobacterium tuberculosis (Mtb) antigen 85 (Ag85) complex, comprising three homologous enzymes required for the biosynthesis of the mycobacterial cell wall. In this study, the Mtb Ag85C enzyme was cocrystallized with azido and adamantyl ebselen derivatives, resulting in two crystallographic structures of 2.01 and 1.30 Å resolution, respectively. Both structures displayed the anticipated covalent modification of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide bond. Continuous difference density for both thiol modifiers allowed for the assessment of interactions that influence ebselen binding and inhibitor orientation that were unobserved in previous Ag85C ebselen structures. The k inact /K I values for ebselen, adamantyl ebselen, and azido ebselen support the importance of observed constructive chemical interactions with Arg239 for increased in vitro efficacy toward Ag85C. To better understand the in vitro kinetic properties of these ebselen derivatives, the energetics of specific protein-inhibitor interactions and relative reaction free energies were calculated for ebselen and both derivatives using density functional theory. These studies further support the different in vitro properties of ebselen and two select ebselen derivatives from our previously published ebselen library with respect to kinetics and protein-inhibitor interactions. In both structures, the α9 helix was displaced farther from the enzyme active site than the previous Ag85C ebselen structure, resulting in the restructuring of a connecting loop and imparting a conformational change to residues believed to play a role in substrate binding specific to Ag85C. These notable structural changes directly affect protein stability, reducing the overall melting temperature by up to 14.5 °C, resulting in the unfolding of protein at physiological temperatures. Additionally, this structural

Physical and biological evaluations of sintered hydroxyapatite/silicone composite with covalent bonding for a percutaneous implant material.

PubMed

Furuzono, Tsutomu; Wang, Pao-Li; Korematsu, Arata; Miyazaki, Kozo; Oido-Mori, Mari; Kowashi, Yusuke; Ohura, Kiyoshi; Tanaka, Junzo; Kishida, Akio

2003-05-15

A composite (HA/silicone) of hydroxyapatite (HA) microparticles with an average diameter of 2.0 micro m covalently linked to a silicone substrate has been developed, and its physical and biological properties as a percutaneous soft-tissue-compatible material have been evaluated. In tensile property measurement, samples of HA/silicone and the original silicone were similar in tensile strength, ca. 7.8 MPa, and elongation at break, ca. 570%. It was found that chemical surface modification with HA particles presented no mechanical disadvantage. In an adhesive-tape peeling test, scanning electron microscopic (SEM) observation showed that HA particles coupled directly to the substrate were not removed. HA particles may bond strongly with the substrate. In human periodontal ligament fibroblast attachment and proliferation experiments, the number of cells attached to HA/silicone was 14 times greater than that attached to the original silicone after 24 h of incubation. The value on HA/silicone was ca. 80% versus that on a tissue-culture plastic used as a positive control. After 72 h of incubation, the number of cells grown on HA/silicone increased to the level of the positive control. In observation of fluorescence microscopy stained by Hoechst 33342, cells appeared to tightly adhere to HA particles coupled to the silicone sheet due to intact nuclear morphology. Observation of cells by fluorescence dye with rhodamin phalloidin showed an extensive F-actin cytoskeleton on HA/silicone. In a 4-week animal implant test, force required to pull out the HA/silicone sheet was 15 times that of the original silicone. HA-particle coating on silicone with covalent linkage gave the inert surface bioactivity. The HA composite thus effectively prevents germ infection percutaneously. Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 65B: 217-226, 2003

Covalently Interlocked Cyclohexa-m-phenylenes and Their Assembly: En Route to Supramolecular 3D Carbon Nanostructures.

PubMed

Dumslaff, Bastian; Reuss, Anna N; Wagner, Manfred; Feng, Xinliang; Narita, Akimitsu; Fytas, George; Müllen, Klaus

2017-08-21

In our search to cluster as many phenylene units as possible in a given space, we have proceeded to the three-dimensional world of benzene-based molecules by employing covalently interlocked cyclohexa-m-phenylenes, as present in the unique paddlewheel-shaped polyphenylene 10. A precursor was conceived, in which freely rotating m-chlorophenylene units provide sufficient solubility along with the necessary proximity for the final ring closure to give 10. Monitoring the assembly of solubilized tert-butyl derivatives of 10 into supramolecular carbon nanostructures by dynamic light scattering (DLS) and Brillouin light scattering (BLS) revealed the dimensions of the initially formed aggregates as well as the amorphous character of the solid state. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Green coconut fiber: a novel carrier for the immobilization of commercial laccase by covalent attachment for textile dyes decolourization.

PubMed

Cristóvão, Raquel O; Silvério, Sara C; Tavares, Ana P M; Brígida, Ana Iraidy S; Loureiro, José M; Boaventura, Rui A R; Macedo, Eugénia A; Coelho, Maria Alice Z

2012-09-01

Commercial laccase formulation was immobilized on modified green coconut fiber silanized with 3-glycidoxypropyltrimethoxysilane, aiming to achieve a cheap and effective biocatalyst. Two different strategies were followed: one point (pH 7.0) and multipoint (pH 10.0) covalent attachment. The influence of immobilization time on enzymatic activity and the final reduction with sodium borohydride were evaluated. The highest activities were achieved after 2 h of contact time in all situations. Commercial laccase immobilized at pH 7.0 was found to have higher activity and higher affinity to the substrate. However, the immobilization by multipoint covalent attachment improved the biocatalyst thermal stability at 50 °C, when compared to soluble enzyme and to the immobilized enzyme at pH 7.0. The Schiff's bases reduction by sodium borohydride, in spite of causing a decrease in enzyme activity, showed to contribute to the increase of operational stability through bonds stabilization. Finally, these immobilized enzymes showed high efficiency in the continuous decolourization of reactive textile dyes. In the first cycle, the decolourization is mainly due to dyes adsorption on the support. However, when working in successive cycles, the adsorption capacity of the support decreases (saturation) and the enzymatic action increases, indicating the applicability of this biocatalyst for textile wastewater treatment.

The SUVR4 Histone Lysine Methyltransferase Binds Ubiquitin and Converts H3K9me1 to H3K9me3 on Transposon Chromatin in Arabidopsis

PubMed Central

Veiseth, Silje V.; Rahman, Mohummad A.; Yap, Kyoko L.; Fischer, Andreas; Egge-Jacobsen, Wolfgang; Reuter, Gunter; Zhou, Ming-Ming; Aalen, Reidunn B.; Thorstensen, Tage

2011-01-01

Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs) on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases) can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3) is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation–dependent and –independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity. PMID:21423664

Understanding RNA modifications: the promises and technological bottlenecks of the ‘epitranscriptome’

PubMed Central

Kapoor, Utkarsh

2017-01-01

The discovery of mechanisms that alter genetic information via RNA editing or introducing covalent RNA modifications points towards a complexity in gene expression that challenges long-standing concepts. Understanding the biology of RNA modifications represents one of the next frontiers in molecular biology. To this date, over 130 different RNA modifications have been identified, and improved mass spectrometry approaches are still adding to this list. However, only recently has it been possible to map selected RNA modifications at single-nucleotide resolution, which has created a number of exciting hypotheses about the biological function of RNA modifications, culminating in the proposition of the ‘epitranscriptome’. Here, we review some of the technological advances in this rapidly developing field, identify the conceptual challenges and discuss approaches that are needed to rigorously test the biological function of specific RNA modifications. PMID:28566301

Covalent Surface Modification of Silicon Oxides with Alcohols in Polar Aprotic Solvents.

PubMed

Lee, Austin W H; Gates, Byron D

2017-09-05

Alcohol-based monolayers were successfully formed on the surfaces of silicon oxides through reactions performed in polar aprotic solvents. Monolayers prepared from alcohol-based reagents have been previously introduced as an alternative approach to covalently modify the surfaces of silicon oxides. These reagents are readily available, widely distributed, and are minimally susceptible to side reactions with ambient moisture. A limitation of using alcohol-based compounds is that previous reactions required relatively high temperatures in neat solutions, which can degrade some alcohol compounds or could lead to other unwanted side reactions during the formation of the monolayers. To overcome these challenges, we investigate the condensation reaction of alcohols on silicon oxides carried out in polar aprotic solvents. In particular, propylene carbonate has been identified as a polar aprotic solvent that is relatively nontoxic, readily accessible, and can facilitate the formation of alcohol-based monolayers. We have successfully demonstrated this approach for tuning the surface chemistry of silicon oxide surfaces with a variety of alcohol containing compounds. The strategy introduced in this research can be utilized to create silicon oxide surfaces with hydrophobic, oleophobic, or charged functionalities.

Covalency in lanthanides. An X-ray absorption spectroscopy and density functional theory study of LnCl6(x-) (x = 3, 2).

PubMed

Löble, Matthias W; Keith, Jason M; Altman, Alison B; Stieber, S Chantal E; Batista, Enrique R; Boland, Kevin S; Conradson, Steven D; Clark, David L; Lezama Pacheco, Juan; Kozimor, Stosh A; Martin, Richard L; Minasian, Stefan G; Olson, Angela C; Scott, Brian L; Shuh, David K; Tyliszczak, Tolek; Wilkerson, Marianne P; Zehnder, Ralph A

2015-02-25

Covalency in Ln-Cl bonds of Oh-LnCl6(x-) (x = 3 for Ln = Ce(III), Nd(III), Sm(III), Eu(III), Gd(III); x = 2 for Ln = Ce(IV)) anions has been investigated, primarily using Cl K-edge X-ray absorption spectroscopy (XAS) and time-dependent density functional theory (TDDFT); however, Ce L3,2-edge and M5,4-edge XAS were also used to characterize CeCl6(x-) (x = 2, 3). The M5,4-edge XAS spectra were modeled using configuration interaction calculations. The results were evaluated as a function of (1) the lanthanide (Ln) metal identity, which was varied across the series from Ce to Gd, and (2) the Ln oxidation state (when practical, i.e., formally Ce(III) and Ce(IV)). Pronounced mixing between the Cl 3p- and Ln 5d-orbitals (t2g* and eg*) was observed. Experimental results indicated that Ln 5d-orbital mixing decreased when moving across the lanthanide series. In contrast, oxidizing Ce(III) to Ce(IV) had little effect on Cl 3p and Ce 5d-orbital mixing. For LnCl6(3-) (formally Ln(III)), the 4f-orbitals participated only marginally in covalent bonding, which was consistent with historical descriptions. Surprisingly, there was a marked increase in Cl 3p- and Ce(IV) 4f-orbital mixing (t1u* + t2u*) in CeCl6(2-). This unexpected 4f- and 5d-orbital participation in covalent bonding is presented in the context of recent studies on both tetravalent transition metal and actinide hexahalides, MCl6(2-) (M = Ti, Zr, Hf, U).

Baseline Chromatin Modification Levels May Predict ...

EPA Pesticide Factsheets

Traditional toxicological paradigms have relied on factors such as age, genotype, and disease status to explain variability in responsiveness to toxicant exposure; however, these are neither sufficient to faithfully identify differentially responsive individuals nor are they modifiable factors that can be leveraged to mitigate the exposure effects. Unlike these factors, the epigenome is dynamic and shaped by an individual's environment. We sought to determine whether baseline levels of specific chromatin modifications correlated with the interindividual variability in their ozone (03)-mediated induction in an air-liquid interface model using primary human bronchial epithelial cells from a panel of 11 donors. We characterized the relationship between the baseline abundance of 6 epigenetic markers with established roles as key regulators of gene expression-histone H3 lysine 4 trimethylation (H3K4me3), H3K27 acetylation (H3K27ac), pan­acetyl H4 (H4ac), histone H3K27 di/trimethylation (H3K27me2/3), unmodified H3, and5-hydroxymethylcytosine (5-hmC)-and the variability in the 03-induced expression of IL-8, IL-6, COX2, and HMOX1. Baseline levels of H3K4me3, H3K27me2/3, and 5-hmC, but not H3K27ac, H4ac, and total H3, correlated with the interindividual variability in 03-mediated induction of HMOX1 and COX2. In contrast, none of the chromatin modifications that we examined correlated with the induction of IL-8 and IL-6. From these findings, we propose an "epigenetic see

Absolute quantitation of NAPQI-modified rat serum albumin by LC-MS/MS: monitoring acetaminophen covalent binding in vivo.

PubMed

LeBlanc, André; Shiao, Tze Chieh; Roy, René; Sleno, Lekha

2014-09-15

Acetaminophen is known to cause hepatoxicity via the formation of a reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), as a result of covalent binding to liver proteins. Serum albumin (SA) is known to be covalently modified by NAPQI and is present at high concentrations in the bloodstream and is therefore a potential biomarker to assess the levels of protein modification by NAPQI. A newly developed method for the absolute quantitation of serum albumin containing NAPQI covalently bound to its active site cysteine (Cys34) is described. This optimized assay represents the first absolute quantitation of a modified protein, with very low stoichiometric abundance, using a protein-level standard combined with isotope dilution. The LC-MS/MS assay is based on a protein standard modified with a custom-designed reagent, yielding a surrogate peptide (following digestion) that is a positional isomer to the target peptide modified by NAPQI. To illustrate the potential of this approach, the method was applied to quantify NAPQI-modified SA in plasma from rats dosed with acetaminophen. The resulting method is highly sensitive (capable of quantifying down to 0.0006% of total RSA in its NAPQI-modified form) and yields excellent precision and accuracy statistics. A time-course pharmacokinetic study was performed to test the usefulness of this method for following acetaminophen-induced covalent binding at four dosing levels (75-600 mg/kg IP), showing the viability of this approach to directly monitor in vivo samples. This approach can reliably quantify NAPQI-modified albumin, allowing direct monitoring of acetaminophen-related covalent binding.

Inactivation of the cloned potassium channel mouse Kv1.1 by the human Kv3.4 'ball' peptide and its chemical modification.

PubMed Central

Stephens, G J; Robertson, B

1995-01-01

1. This study used the whole-cell patch clamp technique to investigate the action of a 28-mer 'inactivation peptide' based on part of the N-terminal sequence of the human Kv3.4 K+ channel (hKv3.4 peptide) on the cloned mouse brain K+ channel mKv1.1 expressed in Chinese hamster ovary (CHO) cells, and compared this with the inactivation produced by Shaker B inactivation peptide (ShB peptide). 2. Inclusion of the hKv3.4 peptide in the patch electrode (320 microM) transformed non-inactivating mKv1.1 into a rapidly inactivating current. The voltage dependence of time constants of decay and steady-state inactivation induced by hKv3.4 peptide were characteristic of an 'A-type' K+ current. 3. The hKv3.4 peptide had no effect on the voltage dependence of activation of mKv1.1, with a mid-point of activation of -8 mV, and a slope factor of 15 mV. Steady-state inactivation curves had a mid-point of inactivation of -36 mV and a slope factor of -7 mV; the time constant of recovery from inactivation at -90 mV was 1.3 s. 4. The chemical modification reagents N-bromoacetamide (NBA, 100 microM) and chloramine-T (CL-T, 500 microM) had no effect on the fast inactivation of mKv1.1 induced by ShB peptide. In contrast, the inactivation caused by hKv3.4 peptide was removed by brief exposure to NBA and CL-T. 5. Chemical modification resulted in a hyperpolarizing shift of -8 mV (CL-T) and -11 mV (NBA) in the voltage dependence of activation of mKv1.1 in the presence of hKv3.4 peptide. 6. Chemical modification was critically dependent on the presence of a cysteine residue at position 6, and not position 24, of hKv3.4 peptide. 7. NBA and CL-T caused only a slight inhibition of unmodified mKv1.1 current with no significant effect on the voltage dependence of mKv1.1 activation, and also had no effect on channel deactivation at -90 mV. 8. Chemical modification experiments were consistent with a selective action on the hKv3.4 peptide itself, specifically at the cysteine residue at position 6

A Cysteine Substitution Probes β3H267 Interactions with Propofol and Other Potent Anesthetics in α1β3γ2L Gamma-Aminobutyric Acid Type A Receptors

PubMed Central

Stern, Alex T.; Forman, Stuart A.

2015-01-01

Background Anesthetic contact residues in γ-aminobutyric acid type A (GABAA) receptors have been identified using photolabels, including two propofol derivatives. O-propofol-diazirine labels H267 in β3 and α1β3 receptors, while m-azi-propofol labels other residues in intersubunit clefts of α1β3. Neither label has been studied in αβγ receptors, the most common isoform in mammalian brain. In αβγ receptors, other anesthetic derivatives photolabel m-azi-propofol labeled residues, but not βH267. Our structural homology model of α1β3γ2L receptors suggests that β3H267 may abut some of these sites. Methods Substituted cysteine modification-protection was used to test β3H267C interactions with four potent anesthetics: propofol, etomidate, alphaxalone, and R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid (mTFD-MPAB). We expressed α1β3γ2L or α1β3H267Cγ2L GABAA receptors in Xenopus oocytes. We used voltage clamp electrophysiology to assess receptor sensitivity to GABA and anesthetics, and to compare para-chloromercuribenzenesulfonate (pCMBS) modification rates with GABA versus GABA plus anesthetics. Results Enhancement of GABA EC5 responses by equi-hypnotic concentrations of all four anesthetics was similar in α1β3γ2L and α1β3H267Cγ2L receptors (n ≥ 3). Direct activation of α1β3H267Cγ2L receptors, but not α1β3γ2L, by mTFD-MPAB and propofol was significantly greater than the other anesthetics. Modification of β3H267C by pCMBS (n ≥ 4) was rapid and accelerated by GABA. Only mTFD-MPAB slowed β3H267C modification (~2-fold; p = 0.011). Conclusions β3H267 in α1β3γ2L GABAA receptors contacts mTFD-MPAB, but not propofol. Our results suggest that β3H267 is near the periphery of one or both transmembrane inter-subunit (α+/β− and γ+/β−) pockets where both mTFD-MPAB and propofol bind. PMID:26569173

Enhanced proton conductivity of Nafion composite membrane by incorporating phosphoric acid-loaded covalent organic framework

NASA Astrophysics Data System (ADS)

Yin, Yongheng; Li, Zhen; Yang, Xin; Cao, Li; Wang, Chongbin; Zhang, Bei; Wu, Hong; Jiang, Zhongyi

2016-11-01

Design and fabrication of efficient proton transport channels within solid electrolytes is crucial and challenging to new energy-relevant devices such as proton exchange membrane fuel cells (PEMFCs). In this study, the phosphoric acid (H3PO4) molecules are impregnated into SNW-1-type covalent organic frameworks (COFs) via vacuum assisted method. High loading of H3PO4 in SNW-1 and low guest leaching rate are achieved due to the similar diameter between H3PO4 and micropores in SNW-1. Then the COF-based composite membranes are fabricated for the first time with impregnated COFs (H3PO4@SNW-1) and Nafion matrix. For the composite membranes, the acid-base pairs formed between H3PO4@SNW-1 networks and Nafion optimize the interfacial interactions and hydrophilic domains. The acidic -PO3H2 groups in pores of H3PO4@SNW-1 provide abundant proton transfer sites. As a result, the continuous proton transfer channels with low energy barrier are created. At the filler content of 15 wt%, the composite membrane exhibits a superior proton conductivity of 0.0604 S cm-1 at 51% relative humidity and 80 °C. At the same time, the maximum power density of single fuel cell is 60.3% higher than that of the recast Nafion membrane.

Covalent Binding Antibodies Suppress Advanced Glycation: On the Innate Tier of Adaptive Immunity

PubMed Central

Shcheglova, T.; Makker, S. P.

2009-01-01

Non-enzymatic protein glycation is a source of metabolic stress that contributes to cytotoxicity and tissue damage. Hyperglycemia has been linked to elevation of advanced glycation endproducts, which mediate much of the vascular pathology leading to diabetic complications. Enhanced glycation of immunoglobulins and their accelerated vascular clearance is proposed as a natural mechanism to intercept alternative advanced glycation endproducts, thereby mitigating microvascular disease. We reported that antibodies against the glycoprotein KLH have elevated reactivity for glycopeptides from diabetic serum. These reactions are mediated by covalent binding between antibody light chains and carbonyl groups of glycated peptides. Diabetic animals that were immunized to induce reactive antibodies had attenuated diabetic nephropathy, which correlated with reduced levels of circulating and kidney-bound glycation products. Molecular analysis of antibody glycation revealed the preferential modification of light chains bearing germline-encoded lambda V regions. We previously noted that antibody fragments carrying V regions in the germline configuration are selected from a human Fv library by covalent binding to a reactive organophosphorus ester. These Fv fragments were specifically modified at light chain V region residues, which map to the combining site at the interface between light and heavy chains. These findings suggest that covalent binding is an innate property of antibodies, which may be encoded in the genome for specific physiological purposes. This hypothesis is discussed in context with current knowledge of the natural antibodies that recognize altered self molecules and the catalytic autoantibodies found in autoimmune disease. PMID:22649604

Photogeneration of singlet oxygen by the phenothiazine derivatives covalently bound to the surface-modified glassy carbon

NASA Astrophysics Data System (ADS)

Blacha-Grzechnik, Agata; Piwowar, Katarzyna; Krukiewicz, Katarzyna; Koscielniak, Piotr; Szuber, Jacek; Zak, Jerzy K.

2016-05-01

The selected group of four amine-derivatives of phenothiazine was covalently grafted to the glassy carbon surface in the four-step procedure consisting of the electrochemical reduction of the diazonium salt followed by the electrochemical and chemical post-modification steps. The proposed strategy involves the bonding of linker molecule to which the photosensitizer is attached. The synthesized organic layers were characterized by means of cyclic voltammetry, XPS and Raman Spectroscopy. It was shown that the phenothiazines immobilized via proposed strategy retain their photochemical properties and are able to generate 1O2 when activated by the laser radiation. The effectiveness of in situ singlet oxygen generation by those new solid photoactive materials was determined by means of UVVis spectroscopy. The reported, covalently modified solid surfaces may find their application as the singlet oxygen photogenerators in the fine chemicals' synthesis or in the wastewater treatment.

Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry.

PubMed

Pesavento, James J; Bullock, Courtney R; LeDuc, Richard D; Mizzen, Craig A; Kelleher, Neil L

2008-05-30

Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >10(4) using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date.

Oxidized Porous Silicon Particles Covalently Grafted with Daunorubicin as a Sustained Intraocular Drug Delivery System

PubMed Central

Chhablani, Jay; Nieto, Alejandra; Hou, Huiyuan; Wu, Elizabeth C.; Freeman, William R.; Sailor, Michael J.; Cheng, Lingyun

2013-01-01

Purpose. To test the feasibility of covalent loading of daunorubicin into oxidized porous silicon (OPS) and to evaluate the ocular properties of sustained delivery of daunorubicin in this system. Methods. Porous silicon was heat oxidized and chemically functionalized so that the functional linker on the surface was covalently bonded with daunorubicin. The drug loading rate was determined by thermogravimetric analysis. Release of daunorubicin was confirmed in PBS and excised rabbit vitreous by mass spectrometry. Daunorubicin-loaded OPS particles (3 mg) were intravitreally injected into six rabbits, and ocular properties were evaluated through ophthalmic examinations and histology during a 3-month study. The same OPS was loaded with daunorubicin using physical adsorption and was evaluated similarly as a control for the covalent loading. Results. In the case of covalent loading, 67 ± 10 μg daunorubicin was loaded into each milligram of the particles while 27 ± 10 μg/mg particles were loaded by physical adsorption. Rapid release of daunorubicin was observed in both PBS and excised vitreous (∼75% and ∼18%) from the physical adsorption loading, while less than 1% was released from the covalently loaded particles. Following intravitreal injection, the covalently loaded particles demonstrated a sustained degradation of OPS with drug release for 3 months without evidence of toxicity; physical adsorption loading revealed a complete release within 2 weeks and localized retinal toxicity due to high daunorubicin concentration. Conclusions. OPS with covalently loaded daunorubicin demonstrated sustained intravitreal drug release without ocular toxicity, which may be useful to inhibit unwanted intraocular proliferation. PMID:23322571

Monobromobimane as an affinity label of the xenobiotic binding site of rat glutathione S-transferase 3-3.

PubMed

Hu, L; Colman, R F

1995-09-15

Monobromobimane (mBBr), besides being a substrate in the presence of glutathione, inactivates rat liver glutathione S-transferase 3-3 at pH 7.5 and 25 degrees C as assayed using 1-chloro-2,4-dinitrobenzene (CDNB). The rate of inactivation is enhanced about 5-fold by S-methylglutathione. Substrate analogs bromosulfophthalein and 2,4-dinitrophenol decrease the rate of inactivation at least 20-fold. Upon incubation for 60 min with 0.25 mM mBBr and S-methylglutathione, the enzyme loses 91% of its activity toward CDNB and incorporates 2.14 mol of reagent/mol of subunit, whereas incubation under the same conditions but with added protectant 2,4-dinitrophenol yields an enzyme that is catalytically active and contains only 0.89 mol of reagent/mol of subunit. mBBR-modified enzyme is fluorescent, and fluorescence energy transfer occurs between intrinsic tryptophan and covalently bound bimane in modified enzyme. Both Tyr115 and Cys114 are modified, but Tyr115 is the initial reaction target and its modification correlates with loss of activity toward CDNB. The fact that the activity toward mBBr is retained by the enzyme after modification suggests that rat isozyme 3-3 has two binding sites for mBBr.

Live imaging of H3K9 acetylation in plant cells

PubMed Central

Kurita, Kazuki; Sakamoto, Takuya; Yagi, Noriyoshi; Sakamoto, Yuki; Ito, Akihiro; Nishino, Norikazu; Sako, Kaori; Yoshida, Minoru; Kimura, Hiroshi; Seki, Motoaki; Matsunaga, Sachihiro

2017-01-01

Proper regulation of histone acetylation is important in development and cellular responses to environmental stimuli. However, the dynamics of histone acetylation at the single-cell level remains poorly understood. Here we established a transgenic plant cell line to track histone H3 lysine 9 acetylation (H3K9ac) with a modification-specific intracellular antibody (mintbody). The H3K9ac-specific mintbody fused to the enhanced green fluorescent protein (H3K9ac-mintbody-GFP) was introduced into tobacco BY-2 cells. We successfully demonstrated that H3K9ac-mintbody-GFP interacted with H3K9ac in vivo. The ratio of nuclear/cytoplasmic H3K9ac-mintbody-GFP detected in quantitative analysis reflected the endogenous H3K9ac levels. Under chemically induced hyperacetylation conditions with histone deacetylase inhibitors including trichostatin A, Ky-2 and Ky-14, significant enhancement of H3K9ac was detected by H3K9ac-mintbody-GFP dependent on the strength of inhibitors. Conversely, treatment with a histone acetyltransferase inhibitor, C646 caused a reduction in the nuclear to cytoplasmic ratio of H3K9ac-mintbody-GFP. Using this system, we assessed the environmental responses of H3K9ac and found that cold and salt stresses enhanced H3K9ac in tobacco BY-2 cells. In addition, a combination of H3K9ac-mintbody-GFP with 5-ethynyl-2′-deoxyuridine labelling confirmed that H3K9ac level is constant during interphase. PMID:28418019

K2 ZnSn3 Se8 : A Non-Centrosymmetric Zinc Selenidostannate(IV) Featuring Interesting Covalently Bonded [ZnSn3 Se8 ]2- Layer and Exhibiting Intriguing Second Harmonic Generation Activity.

PubMed

Zhou, Molin; Jiang, Xingxing; Yang, Yi; Guo, Yangwu; Lin, Zheshuai; Yao, JJiyong; Wu, Yicheng

2017-06-19

Non-centrosymmetric zinc selenidostannate(IV) K 2 ZnSn 3 Se 8 was synthesized. It features interesting covalently bonded [ZnSn 3 Se 8 ] 2- layers with K + cations filling in the interlayer voids. The phonon spectrum was calculated to clarify its structural stability. Based on the X-ray diffraction data along with the Raman spectrum, the major bonding features of the title compound were identified. According to the UV/vis-NIR spectroscopy, K 2 ZnSn 3 Se 8 possesses a typical direct band gap of 2.10 eV, which is in good agreement with the band structure calculations. Moreover, our experimental measurements and detailed theoretical calculations reveal that K 2 ZnSn 3 Se 8 is a new phase-matchable nonlinear optical material with a powder second harmonic generation (SHG) signal about 0.6 times of that of AgGaS 2 . © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, L.I.; Bodwell, J.E.; Mendel, D.B.

1988-05-17

Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. The authors have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel. Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with (/sup 3/H)dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single (/sup 3/H)dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the (/sup 3/H)dexamethasone 21-mesylate was located at position 5 frommore » the amino terminus. Dual-isotope labeling studies with (/sup 3/H)dexamethasone 21-mesylate and (/sup 35/S)methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of (/sup 3/H)dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus. On the basis of the published amino acid sequence of the murine glucocorticoid receptor, their data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate. They have confirmed this finding by demonstrating that a synthetic peptide representing the amino acid sequence 640-650 of the murine glucocorticoid receptor behaves in an identical manner on reversed-phase HPLC as the trypsin-generated peptide from intact cells.« less

Inactivation of chloroplast H(+)-ATPase by modification of Lys beta 359, Lys alpha 176 and Lys alpha 266.

PubMed

Horbach, M; Meyer, H E; Bickel-Sandkötter, S

1991-09-01

Treatment of isolated, latent chloroplast ATPase with pyridoxal-5-phosphate (pyridoxal-P) in presence of Mg2+ causes inhibition of dithiothreitol-activated plus heat-activated ATP hydrolysis. The amount of [3H]pyridoxal-P bound to chloroplast coupling factor 1 (CF1) was estimated to run up to 6 +/- 1 pyridoxal-P/enzyme, almost equally distributed between the alpha- and beta-subunits. Inactivation, however, is complete after binding of 1.5-2 pyridoxal-P/CF1, suggesting that two covalently modified lysines prevent the activation of the enzyme. ADP as well as ATP in presence of Mg2+ protects the enzyme against inactivation and concomittantly prevents incorporation of a part of the 3H-labeled pyridoxal-P into beta- and alpha-subunits. Phosphate prevents labeling of the alpha-subunit, but has only a minor effect on protection against inactivation. The data indicate a binding site at the interface between the alpha- and beta-subunits. Cleavage of the pyridoxal-P-labeled subunits with cyanogen bromide followed by sequence analysis of the labeled peptides led to the detection of Lys beta 359, Lys alpha 176 and Lys alpha 266, which are closely related to proposed nucleotide-binding regions of the alpha- and beta-subunits.

A durable and biocompatible ascorbic acid-based covalent coating method of polydimethylsiloxane for dynamic cell culture.

PubMed

Leivo, Joni; Virjula, Sanni; Vanhatupa, Sari; Kartasalo, Kimmo; Kreutzer, Joose; Miettinen, Susanna; Kallio, Pasi

2017-07-01

Polydimethylsiloxane (PDMS) is widely used in dynamic biological microfluidic applications. As a highly hydrophobic material, native PDMS does not support cell attachment and culture, especially in dynamic conditions. Previous covalent coating methods use glutaraldehyde (GA) which, however, is cytotoxic. This paper introduces a novel and simple method for binding collagen type I covalently on PDMS using ascorbic acid (AA) as a cross-linker instead of GA. We compare the novel method against physisorption and GA cross-linker-based methods. The coatings are characterized by immunostaining, contact angle measurement, atomic force microscopy and infrared spectroscopy, and evaluated in static and stretched human adipose stem cell (hASC) cultures up to 13 days. We found that AA can replace GA as a cross-linker in the covalent coating method and that the coating is durable after sonication and after 6 days of stretching. Furthermore, we show that hASCs attach and proliferate better on AA cross-linked samples compared with physisorbed or GA-based methods. Thus, in this paper, we provide a new PDMS coating method for studying cells, such as hASCs, in static and dynamic conditions. The proposed method is an important step in the development of PDMS-based devices in cell and tissue engineering applications. © 2017 The Author(s).

Regulating the Regulator: Post-Translational Modification of Ras

PubMed Central

Ahearn, Ian M.; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R.

2013-01-01

Ras proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on Ras is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which regulate the activation state of Ras without covalently modifying it. In contrast, post-translational modifications (PTMs) of Ras proteins direct them to various cellular membranes and, in some cases, modulate GTP–GDP exchange. Important Ras PTMs include the constitutive and irreversible remodelling of its C-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications including phosphorylation, peptidyl-proly isomerisation, mono- and di-ubiquitination, nitrosylation, ADP ribosylation and glucosylation. PMID:22189424

Th-Based Endohedral Metallofullerenes: Anomalous Metal Position and Significant Metal-Cage Covalent Interactions with the Involvement of Th 5f Orbitals.

PubMed

Li, Ying; Yang, Le; Liu, Chang; Hou, Qinghua; Jin, Peng; Lu, Xing

2018-05-29

Endohedral metallofullerenes (EMFs) containing actinides are rather intriguing due to potential 5f-orbital participation in the metal-metal or metal-cage bonding. In this work, density functional theory calculations first characterized the structure of recently synthesized ThC 74 as Th@ D 3 h (14246)-C 74 . We found that the thorium atom adopts an unusual off-axis position inside cage due to small metal ion size and the requirement of large coordination number, which phenomenon was further extended to other Th-based EMFs. Significantly, besides the strong metal-cage electrostatic attractions, topological and orbital analysis revealed that all the investigated Th-based EMFs exhibit obvious covalent interactions between metal and cage with substantial contribution from the Th 5f orbitals. The encapsulation by fullerenes is thus proposed as a practical pathway toward the f-orbital covalency for thorium. Interestingly, the anomalous internal position of Th led to a novel three-dimensional metal trajectory at elevated temperatures in the D 3 h -C 74 cavity, as elucidated by the static computations and molecular dynamic simulations.

Histone H3 Lysine Methylation in Cognition and Intellectual Disability Disorders

ERIC Educational Resources Information Center

Parkel, Sven; Lopez-Atalaya, Jose P.; Barco, Angel

2013-01-01

Recent research indicates that epigenetic mechanisms and, in particular, the post-translational modification (PTM) of histones may contribute to memory encoding and storage. Among the dozens of possible histone PTMs, the methylation/demethylation of lysines in the N-terminal tail of histone H3 exhibits particularly strong links with cognitive…

Kinetics of the biodegradation of phenol in wastewaters from the chemical industry by covalently immobilized Trichosporon cutaneum cells.

PubMed

Yotova, Lyubov; Tzibranska, Irene; Tileva, Filadia; Markx, G H; Georgieva, Nelly

2009-03-01

A simple method for the preparation of the biocatalyst with whole cells is presented, and the applicability of the technique for biodegradation of phenol in wastewater from the chemical industries using the basidomycetes yeast Trichosporon cutaneum is explored. Kinetic studies of the influence of other compounds contained in wastewater as naphthalene, benzene, toluene and pyridine indicate that apart from oil fraction, which is removed, the phenol concentration is the only major factor limiting the growth of immobilized cells. Mathematical models are applied to describe the kinetic behavior of immobilized yeast cells. From the analysis of the experimental curves was shown that the obtained values for the apparent rate parameters vary depending on the substrate concentration (mu(maxapp) from 0.35 to 0.09 h(-1) and K (sapp) from 0.037 to 0.4 g dm(-3)). The inhibitory effect of the phenol on the obtained yield coefficients was investigated too. It has been shown that covalent immobilization of T. cutaneum whole cells to plastic carrier beads is possible, and that cell viability and phenol degrading activity are maintained after the chemical modification of cell walls during the binding procedure. The results obtained indicate a possible future application of immobilized T. cutaneum for destroying phenol in industrial wastewaters.

Synthesis and structures of new niobium cluster compounds with pyridinium cations: (PyrH){sub 2}[Nb{sub 6}Cl{sub 18}].EtOH (Pyr: pyridine, Et: ethyl) and the cubic modification of (PyrH){sub 2}[Nb{sub 6}Cl{sub 18}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flemming, Anke; Hoppe, Alessandra; Koeckerling, Martin

2008-10-15

Slow crystallization of (PyrH){sub 2}[Nb{sub 6}Cl{sub 18}] from hot ethanol solution affords triclinic (PyrH){sub 2}[Nb{sub 6}Cl{sub 18}].EtOH. Treatment of [Nb{sub 6}Cl{sub 14}(H{sub 2}O){sub 4}].4H{sub 2}O with pyridine in a methanol solution gives the second title compound, the cubic modification of (PyrH){sub 2}[Nb{sub 6}Cl{sub 18}]. Both structures were determined by single crystal X-ray diffraction, (PyrH){sub 2}[Nb{sub 6}Cl{sub 18}].EtOH: P1-bar , a=9.3475(3), b=9.3957(3), c=10.8600(3) A, {alpha}=82.582(1){sup o}, {beta}=78.608(1){sup o}, and {gamma}=78.085(1){sup o}, Z=1, R{sub 1}(F)/wR{sub 2}(F{sup 2})=0.0254/0.0573, cub.-(PyrH){sub 2}[Nb{sub 6}Cl{sub 18}]: Fd3-bar m, a=19.935(2) A, Z=8, R{sub 1}(F)/wR{sub 2}(F{sup 2})=0.0557/0.1796. The cluster compounds contain isolated, molecular [Nb{sub 6}Cl{sup i}{sub 12}Cl{sup a}{sub 6}]{supmore » 2-} cluster anions with an octahedron of metal atoms edge bridged by chlorido ligands with additional ones on all the six exo positions. These cluster anions are separated by the pyridinium cations and ethanol solvent molecules, respectively. For the cubic modification of (PyrH){sub 2}[Nb{sub 6}Cl{sub 18}], a structural comparison is given to the known rhombohedral modification using the group-subgroup relations as expressed by a Baernighausen tree. - Graphical abstract: The synthesis and structure of a second cubic modification of (PyrH){sub 2}[Nb{sub 6}Cl{sub 18}] and of the new (PyrH){sub 2}[Nb{sub 6}Cl{sub 18}].EtOH are reported, both of which contain isolated niobium halide cluster anions with an octahedral core of metal atoms.« less

Signal transduction in a covalent post-assembly modification cascade

NASA Astrophysics Data System (ADS)

Pilgrim, Ben S.; Roberts, Derrick A.; Lohr, Thorsten G.; Ronson, Tanya K.; Nitschke, Jonathan R.

2017-12-01

Natural reaction cascades control the movement of biomolecules between cellular compartments. Inspired by these systems, we report a synthetic reaction cascade employing post-assembly modification reactions to direct the partitioning of supramolecular complexes between phases. The system is composed of a self-assembled tetrazine-edged FeII8L12 cube and a maleimide-functionalized FeII4L6 tetrahedron. Norbornadiene (NBD) functions as the stimulus that triggers the cascade, beginning with the inverse-electron-demand Diels-Alder reaction of NBD with the tetrazine moieties of the cube. This reaction generates cyclopentadiene as a transient by-product, acting as a relay signal that subsequently undergoes a Diels-Alder reaction with the maleimide-functionalized tetrahedron. Cyclooctyne can selectively inhibit the cascade by outcompeting NBD as the initial trigger. Initiating the cascade with 2-octadecyl NBD leads to selective alkylation of the tetrahedron upon cascade completion. The increased lipophilicity of the C18-tagged tetrahedron drives this complex into a non-polar phase, allowing its isolation from the initially inseparable mixture of complexes.

Covalently Bound Monomolecular Layers on Si Single Crystals

NASA Astrophysics Data System (ADS)

Chidsey, Christopher E. D.

1996-03-01

Methods and reagents borrowed from the molecular synthetic chemistry of silicon compounds have been used to form covalently bound monomolecular layers on silicon single crystals. Organic monolayers bound covalently to silicon could form the basis for silicon/organic interfaces useful in sensor structures. In a representative reaction, alkyl monolayers with densities approaching that of crystalline polyethylene have been prepared by the radical-initiated insertion of 1-alkenes into the Si-H bonds of hydrogen-terminated Si(111) surfaces footnote M. R. Linford, P. Fenter, P. M. Eisenberger and C. E. D Chidsey, J. Am. Chem. Soc. 117, 3145-3155 (1995). It has recently been found that this insertion reaction can also be initiated by illumination with UV light having sufficient energy to break the Si-H bond. Synchrotron-based high-resolution photoelectron spectroscopy and diffraction have demonstrated the expected Si-C bond in such monolayers footnote J. H. Terry, R. Cao, P. A. Pianetta, M. R. Linford and C. E. D. Chidsey, unpublished results. An alternate approach to similar monolayers has been found to be the chlorination of hydrogen-terminated Si(111) with Cl_2, followed by the nucleophilic displacement of chlorine with alkyl lithium reagents. The well-behaved chemical transformations of the hydrogen-terminated silicon surfaces appear to result from the essentially bulk termination of the silicon lattice with closed-shell silicon hydride "functional groups" on the surface. In addition to the formation of novel organic layers, a full understanding of the reactivity of the hydrogen-terminated silicon surfaces should lead to better control of key technological silicon interfaces such as Si/SiO_2, Si/epi-Si, and Si/metal.

Reactive Solute Transport in Streams: 2. Simulation of a pH Modification Experiment

NASA Astrophysics Data System (ADS)

Runkel, Robert L.; McKnight, Diane M.; Bencala, Kenneth E.; Chapra, Steven C.

1996-02-01

We present an application of an equilibrium-based solute transport model to a pH-modification experiment conducted on the Snake River, an acidic, metal-rich stream located in the Rocky Mountains of Colorado. During the experiment, instream pH decreased from 4.2 to 3.2, causing a marked increase in dissolved iron concentrations. Model application requires specification of several parameters that are estimated using tracer techniques, mass balance calculations, and geochemical data. Two basic questions are addressed through model application: (1) What are the processes responsible for the observed increase in dissolved iron concentrations? (2) Can the identified processes be represented within the equilibrium-based transport model? Simulation results indicate that the increase in iron was due to the dissolution of hydrous iron oxides and the photoreduction of ferric iron. Dissolution from the streambed is represented by considering a trace compartment consisting of freshly precipitated hydrous iron oxide and an abundant compartment consisting of aged precipitates that are less soluble. Spatial variability in the solubility of hydrous iron oxide is attributed to heterogeneity in the streambed sediments, temperature effects, and/or variability in the effects of photoreduction. Solubility products estimated via simulation fall within a narrow range (pKsp from 40.2 to 40.8) relative to the 6 order of magnitude variation reported for laboratory experiments (pKsp from 37.3 to 43.3). Results also support the use of an equilibrium-based transport model as the predominate features of the iron and pH profiles are reproduced. The model provides a valuable tool for quantifying the nature and extent of pH-dependent processes within the context of hydrologic transport.

Gene Expression and Chromatin Modifications Associated with Maize Centromeres.

PubMed

Zhao, Hainan; Zhu, Xiaobiao; Wang, Kai; Gent, Jonathan I; Zhang, Wenli; Dawe, R Kelly; Jiang, Jiming

2015-11-12

Centromeres are defined by the presence of CENH3, a variant of histone H3. Centromeres in most plant species contain exclusively highly repetitive DNA sequences, which has hindered research on structure and function of centromeric chromatin. Several maize centromeres have been nearly completely sequenced, providing a sequence-based platform for genomic and epigenomic research of plant centromeres. Here we report a high resolution map of CENH3 nucleosomes in the maize genome. Although CENH3 nucleosomes are spaced ∼190 bp on average, CENH3 nucleosomes that occupied CentC, a 156-bp centromeric satellite repeat, showed clear positioning aligning with CentC monomers. Maize centromeres contain alternating CENH3-enriched and CENH3-depleted subdomains, which account for 87% and 13% of the centromeres, respectively. A number of annotated genes were identified in the centromeres, including 11 active genes that were located exclusively in CENH3-depleted subdomains. The euchromatic histone modification marks, including H3K4me3, H3K36me3 and H3K9ac, detected in maize centromeres were associated mainly with the active genes. Interestingly, maize centromeres also have lower levels of the heterochromatin histone modification mark H3K27me2 relative to pericentromeric regions. We conclude that neither H3K27me2 nor the three euchromatic histone modifications are likely to serve as functionally important epigenetic marks of centromere identity in maize. Copyright © 2016 Zhao et al.

Gene Expression and Chromatin Modifications Associated with Maize Centromeres

PubMed Central

Zhao, Hainan; Zhu, Xiaobiao; Wang, Kai; Gent, Jonathan I.; Zhang, Wenli; Dawe, R. Kelly; Jiang, Jiming

2015-01-01

Centromeres are defined by the presence of CENH3, a variant of histone H3. Centromeres in most plant species contain exclusively highly repetitive DNA sequences, which has hindered research on structure and function of centromeric chromatin. Several maize centromeres have been nearly completely sequenced, providing a sequence-based platform for genomic and epigenomic research of plant centromeres. Here we report a high resolution map of CENH3 nucleosomes in the maize genome. Although CENH3 nucleosomes are spaced ∼190 bp on average, CENH3 nucleosomes that occupied CentC, a 156-bp centromeric satellite repeat, showed clear positioning aligning with CentC monomers. Maize centromeres contain alternating CENH3-enriched and CENH3-depleted subdomains, which account for 87% and 13% of the centromeres, respectively. A number of annotated genes were identified in the centromeres, including 11 active genes that were located exclusively in CENH3-depleted subdomains. The euchromatic histone modification marks, including H3K4me3, H3K36me3 and H3K9ac, detected in maize centromeres were associated mainly with the active genes. Interestingly, maize centromeres also have lower levels of the heterochromatin histone modification mark H3K27me2 relative to pericentromeric regions. We conclude that neither H3K27me2 nor the three euchromatic histone modifications are likely to serve as functionally important epigenetic marks of centromere identity in maize. PMID:26564952

A potent, covalent inhibitor of orotidine 5'-monophosphate decarboxylase with antimalarial activity.

PubMed

Bello, Angelica M; Poduch, Ewa; Fujihashi, Masahiro; Amani, Merhnaz; Li, Yan; Crandall, Ian; Hui, Raymond; Lee, Ping I; Kain, Kevin C; Pai, Emil F; Kotra, Lakshmi P

2007-03-08

Orotidine 5'-monophosphate decarboxylase (ODCase) has evolved to catalyze the decarboxylation of orotidine 5'-monophosphate without any covalent intermediates. Active site residues in ODCase are involved in an extensive hydrogen-bonding network. We discovered that 6-iodouridine 5'-monophosphate (6-iodo-UMP) irreversibly inhibits the catalytic activities of ODCases from Methanobacterium thermoautotrophicum and Plasmodium falciparum. Mass spectral analysis of the enzyme-inhibitor complex confirms covalent attachment of the inhibitor to ODCase accompanied by the loss of two protons and the iodo moiety. The X-ray crystal structure (1.6 A resolution) of the complex of the inhibitor and ODCase clearly shows the covalent bond formation with the active site Lys-72 [corrected] residue. 6-Iodo-UMP inhibits ODCase in a time- and concentration-dependent fashion. 6-Iodouridine, the nucleoside form of 6-iodo-UMP, exhibited potent antiplasmodial activity, with IC50s of 4.4 +/- 1.3 microM and 6.2 +/- 0.7 microM against P. falciparum ItG and 3D7 isolates, respectively. 6-Iodouridine 5'-monophosphate is a novel covalent inhibitor of ODCase, and its nucleoside analogue paves the way to a new class of inhibitors against malaria.

Molecular single-bond covalent radii for elements 1-118.

PubMed

Pyykkö, Pekka; Atsumi, Michiko

2009-01-01

A self-consistent system of additive covalent radii, R(AB)=r(A) + r(B), is set up for the entire periodic table, Groups 1-18, Z=1-118. The primary bond lengths, R, are taken from experimental or theoretical data corresponding to chosen group valencies. All r(E) values are obtained from the same fit. Both E-E, E-H, and E-CH(3) data are incorporated for most elements, E. Many E-E' data inside the same group are included. For the late main groups, the system is close to that of Pauling. For other elements it is close to the methyl-based one of Suresh and Koga [J. Phys. Chem. A 2001, 105, 5940] and its predecessors. For the diatomic alkalis MM' and halides XX', separate fits give a very high accuracy. These primary data are then absorbed with the rest. The most notable exclusion are the transition-metal halides and chalcogenides which are regarded as partial multiple bonds. Other anomalies include H(2) and F(2). The standard deviation for the 410 included data points is 2.8 pm.

Physico-chemical characterization, density functional theory (DFT) studies and Hirshfeld surface analysis of a new organic optical material: 1H-benzo[d]imidazol-3-ium-2,4,6-trinitrobenzene-1,3 bis(olate)

NASA Astrophysics Data System (ADS)

Dhamodharan, P.; Sathya, K.; Dhandapani, M.

2017-10-01

A novel organic crystal, 1H-benzo[d]imidazol-3-ium-2,4,6-trinitrobenzene-1,3 bis(olate) (BITB), was synthesized. Single crystals of BITB were harvested by solution growth-slow evaporation technique. 1H and 13C NMR spectroscopic techniques were utilized to confirm the presence of various types of carbons and protons in BITB. Single crystal XRD confirms that BITB crystallizes in monoclinic system with a space group of P21/n. The suitability of this material for optical applications was assessed by optical absorption, transmittance, reflectance and refractive index spectroscopic techniques. Gaussian 09 program at B3LYP/6-311++G(d,p) level of basis set as used for the optimization of molecular structure of BITB. Greater first order hyperpolarizability value of BITB is due to intensive hydrogen bond network in the crystal. The value is 15 times greater than that of Urea, a reference standard. Computation of frontier molecular orbitals and electrostatic potential surface helped to understand the electron density and reactive sites in BITB. The material was thermally stable up to 220 °C. Hirshfeld surface analysis was performed to quantify the covalent and non covalent interactions.

Covalent immobilization of metal organic frameworks onto chemical resistant poly(ether ether ketone) jacket for stir bar extraction.

PubMed

Wang, Chenlu; Zhou, Wei; Liao, Xiaoyan; Wang, Xuemei; Chen, Zilin

2018-09-26

Preparation of stir bar extraction (SBSE) device with high physical and chemical stability is important and challenging by date. A novel poly (ether ether ketone) (PEEK) tube with excellent mechanical property and chemical stability was firstly used as jacket of metal bar for preparation of stir bar. By employing covalent modification method, the inherent chemical resistant problem of PEEK which restricts the modification of sorbents was well solved. After functionalization, plenty of benzoic acid groups were formed onto the PEEK jacket. Metal organic frameworks of aluminium-based Materials of Institute Lavoisier-68 (MIL-68) was in situ immobilized onto the PEEK surface (MIL-68@PEEK) by the bonding with benzoic acid groups. Afterwards, a facile dumbbell-shaped structure was designed for reducing the friction between sorbents and bottom of container. Due to superior property of the PEEK jacket and the covalent modification method, the MIL-68 modified PEEK jacket SBSE device showed good robustness. After coupling with HPLC-MS/MS, the MIL-68@PEEK-based SBSE device was used to analyse of three parabens including methyl paraben, ethyl paraben and propyl paraben. The method had low limit detection up to 1 pg mL -1 with good linearity (R 2  ≥ 0.9978) and good reproducibility (relative standard deviation ≤ 9.74%). The method has been applied to the detection of parabens in cosmetics and rabbit plasma after painted with cosmetics with recoveries between 73.25% and 104.23%. Copyright © 2018 Elsevier B.V. All rights reserved.

H3K27me3 dynamics dictate evolving uterine states in pregnancy and parturition

PubMed Central

Nancy, Patrice; Siewiera, Johan; Tagliani, Elisa; Osokine, Ivan; Manandhar, Priyanka; Clementi, Caterina

2017-01-01

Uncovering the causes of pregnancy complications such as preterm labor requires greater insight into how the uterus remains in a noncontractile state until term and then surmounts this state to enter labor. Here, we show that dynamic generation and erasure of the repressive histone modification tri-methyl histone H3 lysine 27 (H3K27me3) in decidual stromal cells dictate both elements of pregnancy success in mice. In early gestation, H3K27me3-induced transcriptional silencing of select gene targets ensured uterine quiescence by preventing the decidua from expressing parturition-inducing hormone receptors, manifesting type 1 immunity, and most unexpectedly, generating myofibroblasts and associated wound-healing responses. In late gestation, genome-wide H3K27 demethylation allowed for target gene upregulation, decidual activation, and labor entry. Pharmacological inhibition of H3K27 demethylation in late gestation not only prevented term parturition, but also inhibited delivery while maintaining pup viability in a noninflammatory model of preterm parturition. Immunofluorescence analysis of human specimens suggested that similar regulatory events might occur in the human decidua. Together, these results reveal the centrality of regulated gene silencing in the uterine adaptation to pregnancy and suggest new areas in the study and treatment of pregnancy disorders. PMID:29202469

Extending the working pH of nitrobenzene degradation using ultrasonic/heterogeneous Fenton to the alkaline range via amino acid modification.

PubMed

ElShafei, Gamal M S; Yehia, F Z; Dimitry, O I H; Badawi, A M; Eshaq, Gh

2015-11-01

Oxides of iron, α-Fe2O3 (I), and copper, CuO (II) prepared by usual precipitation method without surfactant were used at room temperature in the process of nitrobenzene (10mgL(-1)) degradation at different pH values with ultrasonic at 20kHz. The degradation was complete in 20 and 30min for (I) and (II), respectively in the pH range 2-7 using1.0gL(-1) of solids and 10mM of H2O2. A remarkable decrease in degradation efficiency was recorded on increasing the pH to values higher than the neutral range. This loss in efficiency was cancelled to a great extent through modifying the used oxides with amino acids. Arginine showed higher improving effect to (II) (1:1 weight ration) than glycine or glutamic acid. Modification of both oxides with increasing amounts of arginine increased the degradation efficiency of (I) in a more regular way than in case of (II). However, the extent of improvement due to amino acid modification was higher in case of (II) because of its originally low degradation efficiency in strongly alkaline media. Copyright © 2014 Elsevier Ltd. All rights reserved.

FOXP3 Orchestrates H4K16 Acetylation and H3K4 Tri-Methylation for Activation of Multiple Genes through Recruiting MOF and Causing Displacement of PLU-1

PubMed Central

Katoh, Hiroto; Qin, Zhaohui S.; Liu, Runhua; Wang, Lizhong; Li, Weiquan; Li, Xiangzhi; Wu, Lipeng; Du, Zhanwen; Lyons, Robert; Liu, Chang-Gong; Liu, Xiuping; Dou, Yali; Zheng, Pan; Liu, Yang

2011-01-01

SUMMARY Both H4K16 acetylation and H3K4 tri-methylation are required for gene activation. However, it is still largely unclear how these modifications are orchestrated by transcriptional factors. Here we analyzed the mechanism of the transcriptional activation by FOXP3, an X-linked suppressor of autoimmune diseases and cancers. FOXP3 binds near transcriptional start sites of its target genes. By recruiting MOF and displacing histone H3K4 demethylase PLU-1, FOXP3 increases both H4K16 acetylation and H3K4 tri-methylation at the FOXP3-associated chromatins of multiple FOXP3-activated genes. RNAi-mediated silencing of MOF reduced both gene activation and tumor suppression by FOXP3, while both somatic mutations in clinical cancer samples and targeted mutation of FOXP3 in mouse prostate epithelial disrupted nuclear localization of MOF. Our data demonstrate a pull-push model in which a single transcription factor orchestrates two epigenetic alterations necessary for gene activation and provide a mechanism for somatic inactivation of the FOXP3 protein function in cancer cells. PMID:22152480

The histone demethylase Jarid1b ensures faithful mouse development by protecting developmental genes from aberrant H3K4me3.

PubMed

Albert, Mareike; Schmitz, Sandra U; Kooistra, Susanne M; Malatesta, Martina; Morales Torres, Cristina; Rekling, Jens C; Johansen, Jens V; Abarrategui, Iratxe; Helin, Kristian

2013-04-01

Embryonic development is tightly regulated by transcription factors and chromatin-associated proteins. H3K4me3 is associated with active transcription and H3K27me3 with gene repression, while the combination of both keeps genes required for development in a plastic state. Here we show that deletion of the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) results in major neonatal lethality due to respiratory failure. Jarid1b knockout embryos have several neural defects including disorganized cranial nerves, defects in eye development, and increased incidences of exencephaly. Moreover, in line with an overlap of Jarid1b and Polycomb target genes, Jarid1b knockout embryos display homeotic skeletal transformations typical for Polycomb mutants, supporting a functional interplay between Polycomb proteins and Jarid1b. To understand how Jarid1b regulates mouse development, we performed a genome-wide analysis of histone modifications, which demonstrated that normally inactive genes encoding developmental regulators acquire aberrant H3K4me3 during early embryogenesis in Jarid1b knockout embryos. H3K4me3 accumulates as embryonic development proceeds, leading to increased expression of neural master regulators like Pax6 and Otx2 in Jarid1b knockout brains. Taken together, these results suggest that Jarid1b regulates mouse development by protecting developmental genes from inappropriate acquisition of active histone modifications.

The Histone Demethylase Jarid1b Ensures Faithful Mouse Development by Protecting Developmental Genes from Aberrant H3K4me3

PubMed Central

Kooistra, Susanne M.; Malatesta, Martina; Morales Torres, Cristina; Rekling, Jens C.; Johansen, Jens V.; Abarrategui, Iratxe; Helin, Kristian

2013-01-01

Embryonic development is tightly regulated by transcription factors and chromatin-associated proteins. H3K4me3 is associated with active transcription and H3K27me3 with gene repression, while the combination of both keeps genes required for development in a plastic state. Here we show that deletion of the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) results in major neonatal lethality due to respiratory failure. Jarid1b knockout embryos have several neural defects including disorganized cranial nerves, defects in eye development, and increased incidences of exencephaly. Moreover, in line with an overlap of Jarid1b and Polycomb target genes, Jarid1b knockout embryos display homeotic skeletal transformations typical for Polycomb mutants, supporting a functional interplay between Polycomb proteins and Jarid1b. To understand how Jarid1b regulates mouse development, we performed a genome-wide analysis of histone modifications, which demonstrated that normally inactive genes encoding developmental regulators acquire aberrant H3K4me3 during early embryogenesis in Jarid1b knockout embryos. H3K4me3 accumulates as embryonic development proceeds, leading to increased expression of neural master regulators like Pax6 and Otx2 in Jarid1b knockout brains. Taken together, these results suggest that Jarid1b regulates mouse development by protecting developmental genes from inappropriate acquisition of active histone modifications. PMID:23637629

Mechanism of histone survival during transcription by RNA polymerase II

PubMed Central

Kulaeva, Olga I

2010-01-01

Transcription of eukaryotic genes by RNA polymerase II is typically accompanied by minimal exchange of histones H3/H4 carrying various covalent modifications. In vitro studies suggest that histone survival is accompanied by the formation of a small transient DNA loop on the surface of the histone octamer including a molecule of transcribing enzyme. PMID:21326897

Kinetics of Electrocatalysis of Dibromoalkyl Reductions Using Electrodes with Covalently Immobilized Metallotetraphenylporphyrins.

DTIC Science & Technology

1981-01-29

Technical Report Using Electrodes with Covalently Immobilized Metal l otetraphenyl porphyri ns G. PERFORMING ORG. REPORT NUMBER 7. AU𔄁IOR(’.) 0...and CH2BrCHBrCH 3 at the surfaces of electrodes to which cobalt(II) or copper (II) tetra(p-aminophenyl)porphyrin has been covalently attached is strongly...27514 ABSTRACT The reduction of PhCHBrCH 2 Br, PhCHBrCHBrPh, and CH2BrCHBrCH3 at the surfaces of electrodes to which cobalt(lI) or copper (If) tetra(p

Time-resolved single-turnover of caa(3) oxidase from Thermus thermophilus. Fifth electron of the fully reduced enzyme converts O(H) into E(H) state.

PubMed

Siletsky, Sergey A; Belevich, Ilya; Belevich, Nikolai P; Soulimane, Tewfik; Verkhovsky, Michael I

2011-09-01

The oxidative part of the catalytic cycle of the caa(3)-type cytochrome c oxidase from Thermus thermophilus was followed by time-resolved optical spectroscopy. Rate constants, chemical nature and the spectral properties of the catalytic cycle intermediates (Compounds A, P, F) reproduce generally the features typical for the aa(3)-type oxidases with some distinctive peculiarities caused by the presence of an additional 5-th redox-center-a heme center of the covalently bound cytochrome c. Compound A was formed with significantly smaller yield compared to aa(3) oxidases in general and to ba(3) oxidase from the same organism. Two electrons, equilibrated between three input redox-centers: heme a, Cu(A) and heme c are transferred in a single transition to the binuclear center during reduction of the compound F, converting the binuclear center through the highly reactive O(H) state into the final product of the reaction-E(H) (one-electron reduced) state of the catalytic site. In contrast to previous works on the caa(3)-type enzymes, we concluded that the finally produced E(H) state of caa(3) oxidase is characterized by the localization of the fifth electron in the binuclear center, similar to the O(H)→E(H) transition of the aa(3)-type oxidases. So, the fully-reduced caa(3) oxidase is competent in rapid electron transfer from the input redox-centers into the catalytic heme-copper site. 2011 Elsevier B.V. All rights reserved.

Influence of pH Adjustment Parameter for Sol-Gel Modification on Structural, Microstructure, and Magnetic Properties of Nanocrystalline Strontium Ferrite

NASA Astrophysics Data System (ADS)

Azis, Raba'ah Syahidah; Sulaiman, Sakinah; Ibrahim, Idza Riati; Zakaria, Azmi; Hassan, Jumiah; Muda, Nor Nadhirah Che; Nazlan, Rodziah; Saiden, Norlaily M.; Fen, Yap Wing; Mustaffa, Muhammad Syazwan; Matori, Khamirul Amin

2018-05-01

Synthesis of nanocrystalline strontium ferrite (SrFe12O19) via sol-gel is sensitive to its modification parameters. Therefore, in this study, an attempt of regulating the pH as a sol-gel modification parameter during preparation of SrFe12O19 nanoparticles sintered at a low sintering temperature of 900 °C has been presented. The relationship of varying pH (pH 0 to 8) on structural, microstructures, and magnetic behaviors of SrFe12O19 nanoparticles were characterized by X-ray diffraction (XRD), field emission scanning microscope (FESEM), and vibrating sample magnetometer (VSM). Varying the pH of precursor exhibited a strong effect on the sintered density, crystal structure and magnetic properties of the SrFe12O19 nanoparticles. As the pH is 0, the SrFe12O19 produced relatively largest density, saturation magnetization, M s, and coercivity, H c, at a low sintering temperature of 900 °C. The grain size of SrFe12O19 is obtained in the range of 73.6 to 133.3 nm. The porosity of the sample affected the density and the magnetic properties of the SrFe12O19 ferrite. It is suggested that the low-temperature sintered SrFe12O19 at pH 0 displayed M s of 44.19 emu/g and H c of 6403.6 Oe, possessing a significant potential for applying in low-temperature co-fired ceramic permanent magnet.

Relationship between covalence and displacive phase transition temperature in RAO{sub 4} and LiAO{sub 3} (R = rare-earth element and A = Nb and Ta)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsunekawa, S.; Fukuda, T.; Kimiyama, T.

Crystal structure analyses by TOF neutron powder diffraction are performed for R TaO{sub 4} (R = rare-earth element) and the Ta-O interatomic distances are determined. The relationship between the covalency of A-O bonds (A = Nb and Ta), which show the most shortening upon phase transition, and the transition temperature is discussed for RAO{sub 4} and LiAO{sub 3}, and the parameters of Ta-O covalence are determined.

THE K-REGION DIHYDRODIOL OF BENZO[A]PYRENE INDUCES DNA DAMAGE AND MORPHOLOGICAL CELL TRANSFORMATION IN C3H10T1/2CL8 MOUSE EMBRYO CELLS WITHOUT THE FORMATION OF DETECTABLE STABLE COVALENT DNA ADDUCTS

EPA Science Inventory

The K -region dihydrodiol ofbenzo[ a ]pyrene induces DNA damage and morphological cell transformation in C3HlOTY2CL8 mouse embryo cells without the formation of detectable stable covalent DNA adducts

Benzo[ a ]pyrene (B[ a ]P) is the most thoroughly studied polycyclic aro...

A highly sensitive immunoassay for atrazine based on covalently linking the small molecule hapten to a urea-glutaraldehyde network on a polystyrene surface.

PubMed

Sai, Na; Sun, Wenjing; Wu, Yuntang; Sun, Zhong; Yu, Guanggui; Huang, Guowei

2016-11-01

A new enzyme-linked immunosorbent assay (ELISA) for atrazine was developed based on covalent bonding of the small molecule hapten, 2-mercaptopropionic acid-4-ethylamino-6-isopropylamino-1,3,5-triazine (MPA-atrazine), to urea-glutaraldehyde (UGA)-treated microtiter plates. In this assay, the microtiter plate surface was treated with the UGA network to both introduce amino groups, which were used to cross-link with the hapten carboxylate groups, and efficiently prevent non-specific adsorption of antibodies, which successfully eliminated the time-consuming routine blocking step. Compared with HNO 3 -H 2 SO 4 -APTES-hapten coated ELISA (modified with a HNO 3 -H 2 SO 4 -APTES mixture and covalent-linked hapten) and conventional ELISA (coated with hapten-carrier protein conjugates), the novel ELISA format increased the sensitivity by approximately 3.5-fold and 7.5-fold, respectively, and saved 2.5h and 34h of coating hapten time, respectively. The method's 50% inhibition concentration for atrazine was 5.54ngmL -1 , and the limit of detection was 0.16ngmL -1 after optimization of reaction conditions. Furthermore, the ELISA was adapted for analysis of atrazine in corn, rice, and water samples, demonstrating recoveries of 90%-108%. Thus, the assay provides a convenient alternative to conventional, laborious immunoassays for routine supervision of residue detection in food and the environment. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA Replication Origin Function Is Promoted by H3K4 Di-methylation in Saccharomyces cerevisiae

PubMed Central

Rizzardi, Lindsay F.; Dorn, Elizabeth S.; Strahl, Brian D.; Cook, Jeanette Gowen

2012-01-01

DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication. PMID:22851644

DNA replication origin function is promoted by H3K4 di-methylation in Saccharomyces cerevisiae.

PubMed

Rizzardi, Lindsay F; Dorn, Elizabeth S; Strahl, Brian D; Cook, Jeanette Gowen

2012-10-01

DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication.

Trafficking and function of the cystic fibrosis transmembrane conductance regulator: a complex network of posttranslational modifications

PubMed Central

McClure, Michelle L.; Barnes, Stephen; Brodsky, Jeffrey L.

2016-01-01

Posttranslational modifications add diversity to protein function. Throughout its life cycle, the cystic fibrosis transmembrane conductance regulator (CFTR) undergoes numerous covalent posttranslational modifications (PTMs), including glycosylation, ubiquitination, sumoylation, phosphorylation, and palmitoylation. These modifications regulate key steps during protein biogenesis, such as protein folding, trafficking, stability, function, and association with protein partners and therefore may serve as targets for therapeutic manipulation. More generally, an improved understanding of molecular mechanisms that underlie CFTR PTMs may suggest novel treatment strategies for CF and perhaps other protein conformational diseases. This review provides a comprehensive summary of co- and posttranslational CFTR modifications and their significance with regard to protein biogenesis. PMID:27474090

Covalent binding of aniline to humic substances. 1. Kinetic studies

USGS Publications Warehouse

Weber, E.J.; Spidle, D.L.; Thorn, K.A.

1996-01-01

The reaction kinetics for the covalent binding of aniline with reconstituted IHSS humic and fulvic acids, unfractionated DOM isolated from Suwannee River water, and whole samples of Suwannee River water have been investigated. The reaction kinetics in each of these systems can be adequately described by a simple second-order rate expression. The effect of varying the initial concentration of aniline on reaction kinetics suggested that approximately 10% of the covalent binding sites associated with Suwannee River fulvic acid are highly reactive sites that are quickly saturated. Based on the kinetic parameters determined for the binding of aniline with the Suwannee River fulvic and humic acid isolates, it was estimated that 50% of the aniline concentration decrease in a Suwannee River water sample could be attributed to reaction with the fulvic and humic acid components of the whole water sample. Studies with Suwannee River fulvic acid demonstrated that the rate of binding decreased with decreasing pH, which parallels the decrease in the effective concentration of the neutral form, or reactive nucleophilic species of aniline. The covalent binding of aniline with Suwannee River fulvic acid was inhibited by prior treatment of the fulvic acid with hydrogen sulfide, sodium borohydride, or hydroxylamine. These observations are consistent with a reaction pathway involving nucleophilic addition of aniline to carbonyl moieties present in the fulvic acid.

Synthesis and characterization of a pH-sensitive conjugate of isoniazid with Fe3O4@SiO2 magnetic nanoparticles.

PubMed

Sedlák, Miloš; Bhosale, Dattatry Shivajirao; Beneš, Ludvík; Palarčík, Jiří; Kalendová, Andrea; Královec, Karel; Imramovský, Aleš

2013-08-15

The Letter describes the preparation and characterization of a conjugate of isoniazid (INH) with magnetic nanoparticles Fe3O4@SiO2 115±60 nm in size. The INH molecules were attached to the surface of nanoparticles by a covalent pH-sensitive amidine bond. The conjugate was characterized by X-ray diffraction, SEM, dynamic light scattering, IR spectroscopy and microanalysis. The conjugate released isoniazid under in vitro conditions (pH=4; 37 °C; t1/2≈115 s). In addition, the cytotoxicity of the Fe3O4@SiO2-INH conjugate was evaluated in SK-BR-3 cells using the xCELLigence system. Copyright © 2013 Elsevier Ltd. All rights reserved.

Interleukin 1 β-induced SMAD2/3 linker modifications are TAK1 dependent and delay TGFβ signaling in primary human mesenchymal stem cells.

PubMed

van den Akker, Guus G; van Beuningen, Henk M; Vitters, Elly L; Koenders, Marije I; van de Loo, Fons A; van Lent, Peter L; Blaney Davidson, Esmeralda N; van der Kraan, Peter M

2017-12-01

Chondrogenic differentiation of mesenchymal stem cells (MSC) requires transforming growth factor beta (TGFβ) signaling. TGFβ binds to the type I receptor activin-like kinase (ALK)5 and results in C-terminal SMAD2/3 phosphorylation (pSMAD2/3C). In turn pSMAD2/3C translocates to the nucleus and regulates target gene expression. Inflammatory mediators are known to exert an inhibitory effect on MSC differentiation. In this study we investigated the effect of interleukin 1 β (IL1β) on SMAD2/3 signaling dynamics and post-translational modifications. Co-stimulation of MSC with TGFβ and IL1β did not affect peak pSMAD2C levels at 1h post-stimulation. Surprisingly, SMAD3 transcriptional activity, as determined by the CAGA 12 -luciferase reporter construct, was enhanced by co-stimulation of TGFβ and IL1β compared to TGFβ alone. Furthermore, IL1β stimulation induced CAGA 12 -luciferase activity in a SMAD dependent way. As SMAD function can be modulated independent of canonical TGFβ signaling through the SMAD linker domain, we studied SMAD2 linker phosphorylation at specific threonine and serine residues. SMAD2 linker threonine and serine modifications were observed within 1h following TGFβ, IL1β or TGFβ and IL1β stimulation. Upon co-stimulation linker modified SMAD2 accumulated in the cytoplasm and SMAD2/3 target gene transcription (ID1, JUNB) at 2-4h was inhibited. A detailed time course analysis of IL1β-induced SMAD2 linker modifications revealed a distinct temperospatial pattern compared to TGFβ. Co-stimulation with both factors resulted in a similar kinetic profile as TGFβ alone. Nevertheless, IL1β did subtly alter TGFβ-induced pSMAD2C levels between 8 and 24h post-stimulation, which was reflected by TGFβ target gene expression (PAI1, JUNB). Direct evidence for the importance of SMAD3 linker modifications for the effect of IL1β on TGFβ signaling was obtained by over-expression of SMAD3 or a SMAD3 linker phospho-mutant. Finally, an inhibitor screening

A First Principles Study of H2 Adsorption on LaNiO3(001) Surfaces

PubMed Central

Pan, Changchang; Chen, Yuhong; Wu, Na; Zhang, Meiling; Yuan, Lihua; Zhang, Cairong

2017-01-01

The adsorption of H2 on LaNiO3 was investigated using density functional theory (DFT) calculations. The adsorption sites, adsorption energy, and electronic structure of LaNiO3(001)/H2 systems were calculated and indicated through the calculated surface energy that the (001) surface was the most stable surface. By looking at optimized structure, adsorption energy and dissociation energy, we found that there were three types of adsorption on the surface. First, H2 molecules completely dissociate and then tend to bind with the O atoms, forming two –OH bonds. Second, H2 molecules partially dissociate with the H atoms bonding to the same O atom to form one H2O molecule. These two types are chemical adsorption modes; however, the physical adsorption of H2 molecules can also occur. When analyzing the electron structure of the H2O molecule formed by the partial dissociation of the H2 molecule and the surface O atom, we found that the interaction between H2O and the (001) surface was weaker, thus, H2O was easier to separate from the surface to create an O vacancy. On the (001) surface, a supercell was constructed to accurately study the most stable adsorption site. The results from analyses of the charge population; electron localization function; and density of the states indicated that the dissociated H and O atoms form a typical covalent bond and that the interaction between the H2 molecule and surface is mainly due to the overlap-hybridization among the H 1s, O 2s, and O 2p states. Therefore, the conductivity of LaNiO3(001)/H2 is stronger after adsorption and furthermore, the conductivity of the LaNiO3 surface is better than that of the LaFeO3 surface. PMID:28772396

Reactive solute transport in streams: 2. Simulation of a pH modification experiment

USGS Publications Warehouse

Runkel, Robert L.; McKnight, Diane M.; Bencala, Kenneth E.; Chapra, Steven C.

1996-01-01

We present an application of an equilibrium-based solute transport model to a pH-modification experiment conducted on the Snake River, an acidic, metal-rich stream located in the Rocky Mountains of Colorado. During the experiment, instream pH decreased from 4.2 to 3.2, causing a marked increase in dissolved iron concentrations. Model application requires specification of several parameters that are estimated using tracer techniques, mass balance calculations, and geochemical data. Two basic questions are addressed through model application: (1) What are the processes responsible for the observed increase in dissolved iron concentrations? (2) Can the identified processes be represented within the equilibrium-based transport model? Simulation results indicate that the increase in iron was due to the dissolution of hydrous iron oxides and the photoreduction of ferric iron. Dissolution from the streambed is represented by considering a trace compartment consisting of freshly precipitated hydrous iron oxide and an abundant compartment consisting of aged precipitates that are less soluble. Spatial variability in the solubility of hydrous iron oxide is attributed to heterogeneity in the streambed sediments, temperature effects, and/or variability in the effects of photoreduction. Solubility products estimated via simulation fall within a narrow range (pKsp from 40.2 to 40.8) relative to the 6 order of magnitude variation reported for laboratory experiments (pKsp from 37.3 to 43.3). Results also support the use of an equilibrium-based transport model as the predominate features of the iron and pH profiles are reproduced. The model provides a valuable tool for quantifying the nature and extent of pH-dependent processes within the context of hydrologic transport.

Flavins as Covalent Catalysts: New Mechanisms Emerge.

PubMed

Piano, Valentina; Palfey, Bruce A; Mattevi, Andrea

2017-06-01

With approximately 1% of proteins being flavoproteins, flavins are at the heart of a plethora of redox reactions in all areas of biology. Thanks to a series of fascinating recent discoveries, in addition to redox chemistry, covalent catalysis is now being recognized more frequently as a common strategy in flavoenzymes, with unprecedented mechanisms becoming apparent. Thus, noncanonical covalent reactions by flavins are emerging as a new pervasive concept in basic enzymology and biochemistry. These diverse enzymes are engaged in most biological processes, positioning the knowledge being gained from these new mechanisms to be translated into drugs that function through covalent mechanisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

Elp3 and Dph3 of Schizosaccharomyces pombe mediate cellular stress responses through tRNALysUUU modifications.

PubMed

Villahermosa, Desirée; Fleck, Oliver

2017-08-03

Efficient protein synthesis in eukaryotes requires diphthamide modification of translation elongation factor eEF2 and wobble uridine modifications of tRNAs. In higher eukaryotes, these processes are important for preventing neurological and developmental defects and cancer. In this study, we used Schizosaccharomyces pombe as a model to analyse mutants defective in eEF2 modification (dph1Δ), in tRNA modifications (elp3Δ), or both (dph3Δ) for sensitivity to cytotoxic agents and thermal stress. The dph3Δ and elp3Δ mutants were sensitive to a range of drugs and had growth defects at low temperature. dph3Δ was epistatic with dph1Δ for sensitivity to hydroxyurea and methyl methanesulfonate, and with elp3Δ for methyl methanesulfonate and growth at 16 °C. The dph1Δ and dph3Δ deletions rescued growth defects of elp3Δ in response to thiabendazole and at 37 °C. Elevated tRNA Lys UUU levels suppressed the elp3Δ phenotypes and some of the dph3Δ phenotypes, indicating that lack of tRNA Lys UUU modifications were responsible. Furthermore, we found positive genetic interactions of elp3Δ and dph3Δ with sty1Δ and atf1Δ, indicating that Elp3/Dph3-dependent tRNA modifications are important for efficient biosynthesis of key factors required for accurate responses to cytotoxic stress conditions.

H3 and H4 Lysine Acetylation Correlates with Developmental and Experimentally Induced Adult Experience-Dependent Plasticity in the Mouse Visual Cortex

PubMed Central

Vierci, Gabriela; Pannunzio, Bruno; Bornia, Natalia; Rossi, Francesco M.

2016-01-01

Histone posttranslational modifications play a fundamental role in orchestrating gene expression. In this work, we analyzed the acetylation of H3 and H4 histones (AcH3–AcH4) and its modulation by visual experience in the mouse visual cortex (VC) during normal development and in two experimental conditions that restore juvenile-like plasticity levels in adults (fluoxetine treatment and enriched environment). We found that AcH3–AcH4 declines with age and is upregulated by treatments restoring plasticity in the adult. We also found that visual experience modulates AcH3–AcH4 in young and adult plasticity-restored mice but not in untreated ones. Finally, we showed that the transporter vGAT is downregulated in adult plasticity-restored models. In summary, we identified a dynamic regulation of AcH3–AcH4, which is associated with high plasticity levels and enhanced by visual experience. These data, along with recent ones, indicate H3–H4 acetylation as a central hub in the control of experience-dependent plasticity in the VC. PMID:27891053