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At least 217 records · Page 12Linked to original sources
PubMed2005

A novel human p53 isoform is an essential element of the ATR-intra-S phase checkpoint.

The archetypal human tumor suppressor p53 is considered to have unique transactivation properties. The assumption is based on the fact that additionally identified human p53 isoforms lack transcriptional activity. However, we provide evidence for the existence of an alternatively spliced p53 isoform (Deltap53) that exerts its transcriptional activity independent from p53. In contrast to p53, Deltap53 transactivates the endogenous p21 and 14-3-3sigma but not the mdm2, bax, and PIG3 promoter. Cell cycle studies showed that Deltap53 displays its differential transcriptional activity only in damaged S phase cells. Upon activation of the ATR-intra-S phase checkpoint, Deltap53, but not p53, transactivates the Cdk inhibitor p21. Induction of p21 results in downregulation of cyclin A-Cdk activity and accordingly attenuation of S phase progression. Data demonstrate that the Deltap53-p21-cyclin A-Cdk pathway is crucial to facilitate uncoupling of repair and replication events, indicating that Deltap53 is an essential element of the ATR-intra-S phase checkpoint.

Alternative Splicing↗
PubMed2006

Different requirements for the association of ATR-ATRIP and 9-1-1 to the stalled replication forks.

DNA replication checkpoint, a surveillance mechanism for S-phase progression, plays a crucial role for the maintenance of genome integrity. A variety of factors have been characterized to be involved in the checkpoint signal transduction. Rpa, a single strand DNA binding protein, was found to be responsible for forming a structure that is recognized by checkpoint sensors and then emits the initial signal for the activation of DNA damage checkpoint. Here we use a mutant of rpa1 gene, rfa1-t11, that has defects in recruiting checkpoint sensor proteins to the site of double strand break, to examine the mutant's effects on the activation of DNA replication checkpoint. We found that the mutant cells activated DNA replication checkpoint normally and showed no defects in recruiting ATR-ATRIP, a major sensor complex that is essential for DNA replication/damage checkpoint, to the site of stalled forks. In contrast, the mutant was defective in recruiting 9-1-1 complex, another sensor complex that functions in DNA damage checkpoint signal transduction, to the stalled forks. Moreover we found that sensitivity for HU obviously appeared in rfa1-t11 mutant when Mrc1 was deleted, while deletion of Rad9, an adaptor specific for damage checkpoint, had subtle effect. These data strongly suggest that rfa1-t11 mutant was mainly defective for activating DNA damage checkpoint and molecular requirement for the recruitment of ATR-ATRIP and 9-1-1 to the stressed forks may be different.

Base Sequence↗
PubMed2005

Surface complexation of condensed phosphate to aluminum hydroxide: an ATR-FTIR spectroscopic investigation.

Two model compounds, sodium pyrophosphate (pyro-P) and sodium tripolyphosphate (tripoly-P), were employed to elucidate the binding mechanisms of condensed phosphate on aluminum hydroxide by utilizing attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Peak assignments for the condensed phosphates in the solution phase and those adsorbed on the surface of aluminum hydroxide were made. Electron delocalization and polarization were employed to explain the peak shifts and the complexation of condensed phosphate with aluminum hydroxide. The tripoly-P and pyro-P were adsorbed on aluminum hydroxide by forming inner-sphere complexes. The adsorbed condensed phosphates were deprotonated in the pH range from 4 to 10. Monodentate, bidentate, and binuclear complexes were formed when pyro-P was adsorbed on aluminum hydroxide, while monodentate and binuclear complexes were formed when tripoly-P was adsorbed. Based on the FTIR data, we proposed that when either bidentate or binuclear complexes were formed, the two oxygen atoms participating in the complexation with aluminum hydroxide could not be originated from the same terminal phosphate moiety. The AlO bond formed in the complexation of pyro-P or tripoly-P with aluminum hydroxide (AlPO(-3)) was not as strong as the HO bond in terminal HPO(-3). The bridging PO(-2) of tripoly-P did not coordinate with aluminum hydroxide. The real-time ATR-FTIR study on condensed phosphate adsorption revealed that a long contact time between condensed phosphates and aluminum hydroxide particles can result in a transformation of an initially formed species into a thermodynamically more stable phase.

Adsorption↗
PubMed2005

Anionic groups on cellulosic fiber surfaces investigated by XPS, FTIR-ATR, and different sorption methods.

Anionic groups (AGs) on different cellulosic fiber surfaces were investigated by methylene blue (MB) and polyelectrolyte (PE) sorption, X-ray photoelectron spectroscopy (XPS), and total attenuated reflectance infrared spectrometry (FTIR-ATR). The MB sorption isotherms fitted well the Langmuir equation that gave consistent estimations of sorption capacities. FTIR-ATR showed that MB molecules had extensive accessibility to the fiber wall pores. Estimation of surface AGs by PE sorption gave much higher values than a new method combining MB sorption and XPS measurements (MB-XPS). The surface AGs in different cellulosic fibers accounted for 1-3% of the total AG content as revealed by MB-XPS. It was suggested that PE molecules can penetrate the fiber wall and form loops or unattached segments at external fiber surfaces that disrupt the PE sorption stoichiometry. The competition of MB and PE for the anionic sites in papermaking was assessed and it was shown that MB ions have a much stronger affinity to AGs than PE molecules.

Adsorption↗
PubMed2005

An ATR-FTIR spectroscopic study of the competitive adsorption between oxalate and malonate at the water-goethite interface.

The competitive adsorption between oxalate and malonate at the water-goethite interface was studied as a function of pH and total ligand concentrations by means of quantitative adsorption measurements and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The results obtained show that ATR-FTIR spectroscopy resolves the individual spectroscopic features of oxalate and malonate when adsorbed simultaneously at the water-goethite interface. The characteristic peaks of all four types of predominating surface complexes existing in the single ligand systems were identified, namely one inner sphere and one outer sphere surface complex for each ligand. The quantitative adsorption data showed that oxalate partially out-competes malonate at the water-goethite interface. Evaluation of the peak area variations as a function of pH indicated that the stronger oxalate adsorption can be ascribed to the more stable inner sphere surface complex of oxalate, which in turn is related to the oxalate five-member chelate ring structure yielding a more stable complex compared to the six-member ring of malonate.

Journal Article↗
PubMed2006

Kinetics of phosphate adsorption on goethite: comparing batch adsorption and ATR-IR measurements.

The adsorption kinetics of phosphate on goethite has been studied by batch adsorption experiments and by in situ ATR-IR spectroscopy at different pH, initial phosphate concentrations and stirring rates. Batch adsorption results are very similar to those reported by several authors, and show a rather fast initial adsorption taking place in a few minutes followed by a slower process taking place in days or weeks. The adsorption kinetics could be also monitored by integrating the phosphate signals obtained in ATR-IR experiments, and a very good agreement between both techniques was found. At pH 4.5 two surface complexes, the bidentate nonprotonated (FeO)(2)PO(2) and the bidentate protonated (FeO)(2)(OH)PO complexes, are formed at the surface. There are small changes in the relative concentrations of these species as the reaction proceeds, and they seem to evolve in time rather independently. At pH 7.5 and 9 the dominating surface species is (FeO)(2)PO(2), which is accompanied by an extra unidentified species at low concentration. They also seem to evolve independently as the reaction proceeds. The results are consistent with a mechanism that involve a fast adsorption followed by a slow diffusion into pores, and are not consistent with surface precipitation of iron phosphate.

Adsorption↗
PubMed2004

ATR-IR spectroscopy as applied to nucleic acid films.

For the first time the ATR technique was applied to obtain IR absorption spectra of DNA and RNA dry films. There was worked out procedure of the nucleic acid removal from germanium plate, which obviously was a main obstacle to application of ATR-IR spectroscopy to nucleic acids. This technique of IR spectroscopy was applied to confirmation of RNA tropism of aurin tricarboxylic acid observed by molecular biological methods.

Bacteriophage lambda↗
PubMed2006

Cell cycle-dependent DNA damage signaling induced by ICRF-193 involves ATM, ATR, CHK2, and BRCA1.

Topoisomerase II is essential for cell proliferation and survival and has been a target of various anticancer drugs. ICRF-193 has long been used as a catalytic inhibitor to study the function of topoisomerase II. Here, we show that ICRF-193 treatment induces DNA damage signaling. Treatment with ICRF-193 induced G2 arrest and DNA damage signaling involving gamma-H2AX foci formation and CHK2 phosphorylation. DNA damage by ICRF-193 was further demonstrated by formation of the nuclear foci of 53BP1, NBS1, BRCA1, MDC1, and FANCD2 and increased comet tail moment. The DNA damage signaling induced by ICRF-193 was mediated by ATM and ATR and was restricted to cells in specific cell cycle stages such as S, G2, and mitosis including late and early G1 phases. Downstream signaling of ATM and ATR involved the phosphorylation of CHK2 and BRCA1. Altogether, our results demonstrate that ICRF-193 induces DNA damage signaling in a cell cycle-dependent manner and suggest that topoisomerase II might be essential for the progression of the cell cycle at several stages including DNA decondensation.

Antineoplastic Agents↗
PubMed2007

Analyzing the ATR-mediated checkpoint using Xenopus egg extracts.

Our knowledge of cell cycle events such as DNA replication and mitosis has been advanced significantly through the use of Xenopus egg extracts as a model system. More recently, Xenopus extracts have been used to investigate the cellular mechanisms that ensure accurate and complete duplication of the genome, processes otherwise known as the DNA damage and replication checkpoints. Here we describe several Xenopus extract methods that have advanced the study of the ATR-mediated checkpoints. These include a protocol for the preparation of nucleoplasmic extract (NPE), which is a soluble extract system useful for studying nuclear events such as DNA replication and checkpoints. In addition, we describe several key assays for studying checkpoint activation as well as methods for using small DNA structures to activate ATR.

Animals↗
PubMed2002

Spectroscopy of polymer/drug formulations processed with supercritical fluids: in situ ATR-IR and Raman study of impregnation of ibuprofen into PVP.

In situ ATR (attenuated total reflectance)-IR spectroscopy has been used to study poly(vinylpyrrolidone) (PVP) films subjected to a solution of ibuprofen in supercritical CO2. The process of impregnation of ibuprofen into PVP has been monitored in situ. It has been shown that the supercritical fluid impregnation process results in ibuprofen being molecularly dispersed in a polymer matrix with ibuprofen molecules interacting with the C=O group of PVP. Raman spectra of ibuprofen impregnated into PVP from supercritical fluid solution have also been measured and compared with the Raman spectra of crystalline ibuprofen. ATR-IR spectroscopy has also revealed specific interactions between the C=O groups of PVP and CO2. Impregnation of ibuprofen into PVP makes the C=O groups of PVP less available for interactions with CO2. It has also been demonstrated that the presence of ibuprofen in PVP also affects sorption of water into PVP.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2005

ATR-FT-IR membrane-based sensor for integrated microliquid-liquid extraction and detection.

A novel straightforward membrane-based sensor, which uses attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy has been developed. The flow cell designed permits the on-line microliquid-liquid extraction of the target analyte into a organic solvent layer (OSL), which was deposited on the ATR surface using a sequential injection manifold. The aqueous and organic phases are separated via a commercial hydrophobic membrane placed on the PTFE piece of the cell. The main advantage of the proposed device is that the OSL can be created and regenerated in a continuous manner using the automatic manifold without opening the cell. The analytes are enriched into the OSL after diffusion through the membrane, which excludes the typical absorption bands of water. In addition, the behavior of different organic solvents was evaluated in order to increase the applicability and versatility of the proposed system. Finally, the analytical performance of the design was established for the detection and quantitation of Triton X100 in water.

Journal Article↗
PubMed2006

Determination of the structure and orientation of organic molecules tethered to flat graphitic carbon by ATR-FT-IR and Raman spectroscopy.

Mono- and multilayers of nitroazobenzene (NAB), azobenzene (AB), nitrobiphenyl (NBP), biphenyl (BP), and fluorene (FL) were covalently bonded to flat pyrolyzed photoresist films (PPF) by electrochemical reduction of their diazonium derivatives. The structure and orientation of the molecular layers were probed with ATR-FT-IR and Raman spectroscopy. A hemispherical germanium ATR element used with p-polarized light at 65 degrees incidence angle yielded high signal/noise IR spectra for monolayer coverage of molecules on PPF. The IR spectra are dominated by in-plane vibrational modes in the 1000-2000-cm(-1) spectral range but also exhibit weaker out-of-plane deformations in the 650-1000-cm(-1) region. The average tilt angle with respect to the surface normal for the various molecules varied from 31.0 +/- 4.5 degrees for NAB to 44.2 +/- 5.4 degrees for FL with AB, NBP, and BP exhibiting intermediate adsorption geometries. Raman intensity ratios of NAB and AB for p- and s-polarized incident light support the conclusion that the chemisorbed molecules are in a predominantly upright orientation. The results unequivocally indicate that molecules electroreduced from their diazonium precursors are not chemisorbed flat on the PPF surface, but rather at an angle, similar to the behavior of Au/thiol self-assembled monolayers, Langmuir-Blodgett films, and porphyrin molecules chemisorbed thermally on silicon and PPF from alkyne and alkene precursors.

Azo Compounds↗
PubMed1995

Site-directed isotope labeling and ATR-FTIR difference spectroscopy of bacteriorhodopsin: the peptide carbonyl group of Tyr 185 is structurally active during the bR-->N transition.

The largest secondary structural change occurs in the bacteriorhodopsin (bR) photocycle during the M-->N transition. In this work site-directed isotope labeling (SDIL) and attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy were used to investigate this conformational change. L-Tyrosine containing a 13C isotope at the carbonyl carbon was selectively incorporated at Tyr 57, Tyr 147, and Tyr 185 by SDIL. This involves the cell-free expression of bR in the presence of Escherichia coli suppressor tRNA(CUATyr) aminoacylated with L-[1-13C]Tyr. ATR-FTIR difference spectroscopy reveals that of the 11 tyrosines, only the peptide carbonyl group of Tyr 185 undergoes a significant structural change during the bR-->N transition. Along with other spectroscopic evidence, this result suggests that the Tyr 185-Pro 186 region of the protein is structurally active and may function as a hinge which facilitates the tilt of the cytoplasmic portion of the F-helix in bacteriorhodopsin during the M-->N transition.

Bacteriorhodopsins↗
PubMed1992

Fluorescence, CD, attenuated total reflectance (ATR) FTIR, and 13C NMR characterization of the structure and dynamics of synthetic melittin and melittin analogues in lipid environments.

The structure and dynamics of synthetic melittin (MLT) and MLT analogues bound to monomyristoylphosphatidylcholine micelles, dimyristoylphosphatidylcholine vesicles, and diacylphosphatidylcholine films have been investigated by fluorescence, CD, attenuated total reflectance (ATR) FTIR, and 13C NMR spectroscopy. All of these methods provide information about peptide secondary structure and/or about the environment of the single tryptophan side chain in these lipid environments. ATR-FTIR data provide additional information about the orientation of helical peptide segments with respect to the bilayer plane. Steady-state fluorescence anisotropy, fluorescence lifetime, and 13C NMR relaxation data are used in concert to provide quantitative information about the dynamics of a single 13C-labeled tryptophan side chain at position 19 in lipid-bound MLT, and at positions 17, 11, and 9, respectively, in lipid-bound MLT analogues. Peptide chain dynamics are probed by NMR relaxation studies of 13C alpha-labeled glycine incorporated into each of the MLT peptides at position 12. The cumulative structural and dynamic data are consistent with a model wherein the N-terminal alpha-helical segment of these peptides is oriented perpendicular to the bilayer plane. Correlation times for the lysolipid-peptide complexes provide evidence for binding of a single peptide monomer per micelle. A model for the membranolytic action of MLT and MLT-like peptides is proposed.

Circular Dichroism↗
PubMed2001

ATR-FTIR study of the structure and orientation of transmembrane domains of the Saccharomyces cerevisiae alpha-mating factor receptor in phospholipids.

The structures of seven synthetic transmembrane domains (TMDs) of the alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae were studied in phospholipid multilayers by transmission Fourier transform infrared (FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopies. Peptide conformation assumed in multilayers depended on the method of sample preparation. Amide proton H/D exchange experiments showed that 60-80% of the NH bonds in these TMDs did not exchange with bulk water in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilayers. FTIR results showed that peptides corresponding to TMDs one, two, and seven were mostly alpha-helical in DMPC multilayers. Peptides corresponding to TMDs three and six assumed predominantly beta-sheet structures, whereas those corresponding to TMDs four and five were a mixture of alpha-helices and beta-sheets. ATR-FTIR showed that in DMPC the alpha-helices of TMDs two and five oriented with tilt angles of 34 degrees and 32 degrees, respectively, with respect to the multilayer normal. Similar results were obtained for six of the transmembrane domains in DMPC/DMPG (4:1) multilayers. In a mixture [POPC/POPE/POPS/PI/ergosterol (30:20:5:20:25)] which mimicked the lipid composition of the S. cerevisiae cell membrane, the percentage of alpha-helical structures found for TMDs one and five increased compared to those in DMPC and DMPC/DMPG (4:1) multilayers, and TMD six exhibited a mixture of beta-sheet ( approximately 60%) and alpha-helical ( approximately 40%) structure. These experiments provide biophysical evidence that peptides representing the seven transmembrane domains in Ste2p assume different structures and tilt angles within a membrane multilayer.

Amides↗
PubMed1999

2D-NMR and ATR-FTIR study of the structure of a cell-selective diastereomer of melittin and its orientation in phospholipids.

Melittin, a 26 residue, non-cell-selective cytolytic peptide, is the major component of the venom of the honey bee Apis mellifera. In a previous study, a diastereomer ([D]-V(5,8),I(17),K(21)-melittin, D-amino acids at positions V(5,8),I(17),K(21)) of melittin was synthesized and its function was investigated [Oren, Z., and Shai, Y. (1997) Biochemistry 36, 1826-1835]. [D]-V(5,8),I(17),K(21)-melittin lost its cytotoxic effects on mammalian cells; however, it retained antibacterial activity. Furthermore, [D]-V(5,8),I(17),K(21)-melittin binds strongly and destabilizes only negatively charged phospholipid vesicles, in contrast to native melittin, which binds strongly also zwitterionic phospholipids. To understand the differences in the properties of melittin and its diastereomer, 2D-NMR experiments were carried out with [D]-V(5,8),I(17),K(21)-melittin, and polarized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy experiments were done with both melittin and [D]-V(5,8), I(17),K(21)-melittin. The structure of the diastereomer was characterized by NMR in water, as well as in three different membrane-mimicking environment, 40% 2,2,2-trifluoroethanol (TFE)/water, methanol, and dodecylphosphocholine/phosphatidylglycerol (DPC/DMPG) micelles. The NMR data revealed an amphipathic alpha-helix only in the C-terminal region of the diastereomer in TFE/water and methanol solutions and in DPC/DMPG micelles. ATR-FTIR experiments revealed that melittin and [D]-V(5,8),I(17),K(21)-melittin are oriented parallel to the membrane surface. This study indicates the role of secondary structure formation in selective cytolytic activity of [D]-V(5,8), I(17),K(21)-melittin. While the N-terminal helical structure is not required for the cytolytic activity toward negatively charged membranes and bacterial cells, it appears to be a crucial structural element for binding and insertion into zwitterionic membranes and for hemolytic activity.

Amino Acid Sequence↗
PubMed2006

Molecular recognition between protein and nicotinamide dinucleotide in intact, proton-translocating transhydrogenase studied by ATR-FTIR Spectroscopy.

Nicotinamide dinucleotide binding to transhydrogenase purified from Escherichia coli was investigated by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Detergent-free transhydrogenase was deposited as a thin film on an ATR prism, and spectra were recorded during perfusion with buffers in the presence and absence of dinucleotide (NADP(+), NADPH, NAD(+), or NADH) in both H(2)O and D(2)O media. IR spectral changes were attributable to the bound dinucleotides and to changes in the protein itself. The dissociation constant of NADPH was estimated to be approximately 5 muM from a titration of the magnitude of the IR changes against the nucleotide concentration. IR spectra of related model compounds were used to assign principle bands of the dinucleotides. This information was combined with IR data on amino acids and with protein crystallographic data to identify interactions between specific parts of the dinucleotides and their binding sites in the protein. Several IR bands of bound nucleotide were sharpened and/or shifted relative to those in aqueous solution, reflecting a restriction to motion and a change in environment upon binding. Alterations in the protein secondary structure indicated by amide I/II changes were distinctly different for NADP(H) and for NAD(H) binding. The data suggest that NADP(H) binding leads to perturbation of a deeply buried part of the polypeptide backbone and to protonation of a carboxylic acid residue.

Deuterium Exchange Measurement↗
PubMed2005

ATR-FT/IR study on the interactions between gliadins and dextrin and their effects on protein secondary structure.

The effects of heat treatment and dextrin addition on the secondary structure of gliadins were investigated by means of attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT/IR). Gliadins and gliadin/dextrin mixtures (before and after thermal treatment) were prepared as a dried protein film on the ATR-FT/IR zinc selenide cell plate and equilibrated at a water activity (a(w)) of 0.06. The results show that gliadins undergo conformational changes upon thermal treatment both in the absence and in the presence of dextrin. In particular, in the thermally treated gliadins, the decrease of the band at around 1651 cm(-)(1) and the increase of the bands at around 1628 and 1690 cm(-)(1) suggest a loss of alpha-helix structure and a higher content of protein aggregates. The same trend was observed in the presence of dextrin. Concerning the interactions between gliadins and dextrin, gliadin/dextrin mixtures show variations in the amide I region compared to native gliadins (e.g., an increase of the band at 1645 cm(-)(1) and the absence of the band at around 1668 cm(-)(1)) that might be due to hydrogen bond formation between gliadins and dextrin. It was also found that the spectrum of gliadin/dextrin mixtures was less affected by the hydration state than that of native gliadins, as observed from the differential spectra obtained by subtraction of the spectrum obtained at a(w) = 0.06 (driest condition tested) from the spectrum of the sample equilibrated at a(w) = 0.84. This could be due to the fact that C=O and N-H groups of gliadins are engaged to form hydrogen bonds with the hydroxyl groups of dextrin, and so they are not perturbed by the presence of water molecules. Finally, water activity effects on the secondary structure of gliadins are also discussed.

Dextrins↗