Fragmentation Step Research Articles

The role of Aurora-A kinase in the Golgi-dependent control of mitotic entry

During mitosis, the Golgi complex undergoes a multi-step fragmentation process that is instrumental to its correct partitioning into the daughter cells. To prepare for this segregation, the Golgi ribbon is initially separated into individual stacks during the G2 phase of the cell cycle. Then, at the onset of mitosis, these individual stacks are further disassembled into dispersed fragments. Inhibition of this Golgi fragmentation step results in a block or delay of G2/M transition, depending on the experimental approach. Thus, correct segregation of the Golgi complex appears to be monitored by a 'Golgi mitotic checkpoint'. Using a microinjection-based approach, we recently identified the first target of the Golgi checkpoint, whereby a block of this Golgi fragmentation impairs recruitment of the mitotic kinase Aurora-A to, and its activation at, the centrosomes. Overexpression of Aurora-A can override this cell cycle block, indicating that Aurora-A is a major effector of the Golgi checkpoint. We have also shown that this block of Aurora-A recruitment to the centrosomes is not mediated by the known mechanisms of regulation of Aurora-A function. Here we discuss our findings in relation to the known functions of Aurora-A.

Mitotic Golgi vesiculation involves mechanisms independent of Ser25 phosphorylation of GM130

During mitosis, the Golgi undergoes two sequential fragmentation steps to break from ribbon to individual stacks, then from stacks to vesicles. While the mechanism that regulates the first step has been studied, it remains obscure how the second vesiculation step is regulated. It has been suggested that Cdk1-dependent phosphorylation of the cis-Golgi matrix protein GM130 regulates the second step. Here we have tested if phorphorylation of GM130 by Cdk1 is required for Golgi vesiculation and mitotic progression. Inhibition of Cdk1 caused a failure of Golgi vesiculation and defects in chromosome congression/segregation. Expression of non-phosphorylatable mutant of GM130 (GM130S25A) in cells depleted of endogenous GM130 caused no apparent defects in Golgi vesiculation and mitotic progression. Similarly, no apparent defects in Golgi vesiculation and mitotic progression were observed when GM130S25A was expressed in GM130-deficient CHO cells. Our observations suggest that while Cdk1 based phosphorylation is essential for mitotic Golgi vesiculation, mammalian cells could possess redundant, S25 phosphorylation of GM130 independent pathways that ensure Golgi vesiculation and mitotic progression.

ChemInform Abstract: An Approach to the Aromadendrane Carbon Skeleton by a Radical Fragmentation/3-exo-Trig Cyclization Sequence.

Abstract A radical fragmentation/cyclization sequence of a [2+2] photoadduct derivative leads to the formation of the aromadendrane ring system. The 5,7 fused rings of this sesquiterpenoid family are formed in the fragmentation step and the cyclopropane moiety is the result of a 3-exo-trig cyclization. The cyclization step is facilitated by the use of SmI2 as the electron donor and by an ester group to stabilize the resultant strained intermediate.

Optimizing immunoslot blot assays and application to low DNA adduct levels using an amplification approach

Immunoslot blot assays have been used for the analysis of many DNA adducts, but problems are frequently encountered in achieving reproducible results. Each step of the assay was examined systematically, and it was found that the major problems are in the DNA fragmentation step and the use of the manifold apparatus. Optimization was performed on both the malondialdehyde–deoxyguanosine (M1dG) adduct and the O6-carboxymethyl–deoxyguanosine (O6CMdG) adduct to demonstrate the applicability to other DNA adducts. Blood samples from the European Prospective Investigation on Cancer (EPIC) study (n=162) were analyzed for M1dG adducts, and the data showed no correlation with adduct levels in other tissues, indicating that the EPIC blood samples were not useful for studying M1dG adducts. Blood samples from a processed meat versus vegetarian diet intervention (n=6) were analyzed for O6CMdG, and many were below the limit of detection. The reduction of background adduct levels in standard DNA was investigated using chemical and whole genome amplification approaches. The latter gave a sensitivity improvement of 2.6 adducts per 107 nucleotides for the analysis of O6CMdG. Subsequent reanalysis for O6CMdG showed a weakly significant increase in O6CMdG on the processed meat diet compared with the vegetarian diet, demonstrating that further studies are warranted.

One-pot asymmetric cyclocarbohydroxylation sequence for the enantioselective synthesis of functionalised cyclopentanes

A new method has been developed for the enantioselective synthesis of highly functionalised cyclopentanes bearing up to three stereogenic centres with very high stereoselectivity. This one-pot process combines an enantioselective organocatalytic Michael addition with a highly diastereoselective [3+2]-cycloaddition-fragmentation step.

Dissolution mechanisms of wood cellulose fibres in NaOH–water

Four wood pulps and a microcrystalline cellulose were dissolved in a NaOH 8%–water solution. Insoluble fractions and clear solution fractions were isolated by centrifugation and were observed by optical microscopy and transmission electron microscopy. Molecular weight distribution, carbohydrate composition and cellulose II content were measured. The dissolution of wood cellulose fibres in NaOH 8%–water solutions occurs by successive dismantlement and fragmentation steps governed by the swelling and the shearing of the original structure. The cellulose from insoluble and clear solution fractions is in both case converted in cellulose II and the insoluble fractions contain embedded mannans. Besides, the molecular weight distributions of cellulose from insoluble and clear solution fractions reveal the existence of heterogeneities in dissolution capacity of the cellulose chains, independent to the degree of polymerization, which are related to the chemical environment of the chains in the fibre structure.

Theoretical evaluation of the order of reactivity of transfer agents utilized in RAFT polymerization

Density functional theory, DFT, PBE1PBE functional and 6-31+G(d,p) basis set in Gaussian 03 software were used in order to determine the reactivity order of the R group in RAFT agents used in the radical addition-fragmentation transfer polymerization, through the evaluation of reactivity parameters such as: global and local electronegativity, hardness, softness, and philicity. It was found that the reactivity order is governed by both the number and the composition of the substituent group (primary, secondary or tertiary); that is, the larger those parameters are the larger factors like steric hindrance, polar effects and electronic interchanges are, which favors the breaking of the C-S bond from the adduct radical, permitting the exit of the leaving radical and allowing, as a consequence, the fragmentation step in RAFT polymerization. Trisubstituted dithioesters with structure S = C(Z)S-R, where Z = Phenyl and R = C(CH(3))(2)CONH(2), C(CH3)2Ph or (CH(3))(2)C(6)H(8)OCH(3), in accordance with the previously exposed, presented the most favorable reactivity parameters.

Small Angle X-ray Scattering Analysis of Deoxyguanosine 5′-Monophosphate Self-Assembing in Solution: Nucleation and Growth of G-Quadruplexes

To solve details of the self-assembling process of guanosine in diluted aqueous solution and to derive a thermodynamical model for quadruplex formation, the structural behavior of deoxyguanosine 5'-monophosphate has been analyzed by in-solution small angle X-ray scattering. The experiments have been performed as a function of guanosine concentration and at fixed guanosine concentration but in the presence of varying amounts of KCl. As a result, the self-assembling process, in terms of both aggregate particle fractions and aggregate length, has been observed to be strongly dependent on composition and largely affected by excess potassium ions in the solution. In particular, the different aggregate forms have been resolved and their concentration derived as a function of sample composition. In accordance with a hierarchical aggregation process, a nucleation and elongation mechanism has been used to derive the thermodynamical parameters for self-assembling. The results show that the annealing and fragmentation steps play an important role in the aggregation process.

An integrated experimental and theoretical investigation on Cu(hfa)2·TMEDA: structure, bonding and reactivity

The physico-chemical properties of the beta-diketonate diamine Cu(ii) compound with hfa (1,1,1,5,5,5-hexafluoro-2-4-pentanedionate) and TMEDA (N,N,N',N' tetramethylethylenediamine), Cu(hfa)(2).TMEDA, have been thoroughly investigated via an integrated multi-technique experimental-computational approach. In the newly found orthorhombic polymorph, as revealed by low temperature single-crystal X-ray studies, the complex is present as a monomer with a distorted octahedral geometry at the Cu(ii) centre. The compound sublimates, without premature side decompositions, at 343 K and 10(-3) Torr. The structural, vibrational, electronic and thermal behavior of the neutral Cu(hfa)(2).TMEDA complex has been investigated along with its fragmentation pathways, initiated by the release of an anionic hfa ligand with formation of a positive Cu(hfa).TMEDA(+) ion. Joint experimental and theoretical analyses led to the rationalization of the first fragmentation steps in terms of the Cu(ii)-ligands bonding properties and Jahn-Teller distortion. The present study suggests applications of Cu(hfa)(2).TMEDA as a precursor for copper and copper oxide materials by Chemical Vapor Deposition.

Glycan family analysis for deducing N-glycan topology from single MS

In the past few years, mass spectrometry (MS) has emerged as the premier tool for identification and quantification of biological molecules such as peptides and glycans. There are two basic strategies: single-MS, which uses a single round of mass analysis, and MS/MS (or higher order MS(n)), which adds one or more additional rounds of mass analysis, interspersed with fragmentation steps. Single-MS offers higher throughput, broader mass coverage and more direct quantitation, but generally much weaker identification. Single-MS, however, does work fairly well for the case of N-glycan identification, which are more constrained than other biological polymers. We previously demonstrated single-MS identification of N-glycans to the level of 'cartoons' (monosaccharide composition and topology) by a system that incorporates an expert's detailed knowledge of the biological sample. In this article, we explore the possibility of ab initio single-MS N-glycan identification, with the goal of extending single-MS, or primarily-single-MS, identification to non-expert users, novel conditions and unstudied tissues. We propose and test three cartoon-assignment algorithms that make inferences informed by biological knowledge about glycan synthesis. To test the algorithms, we used 71 single-MS spectra from a variety of tissues and organisms, containing more than 2800 manually annotated peaks. The most successful of the algorithms computes the most richly connected subgraph within a 'cartoon graph'. This algorithm uniquely assigns the correct cartoon to more than half of the peaks in 41 out of the 71 spectra.

Methylaminosilane fed Inductively Coupled Plasmas for Silicon Nitride Deposition

AbstractSilicon nitride‐like films can be deposited by inductively coupled discharges fed with dimethylaminosilanes without addition of reactive gases. At high input power fairly inorganic films can be obtained. In the present work in situ diagnostics of plasma phase, i.e. Fourier transform infrared spectroscopy (FT‐IRAS) and optical emission spectroscopy (OES), have been utilized to investigate the deposition mechanism of bis(dimethylamino)dimethylsilane (BDMADMS) fed discharges. Results indicate that the monomer undergoes a pronounced depletion even at low input power and that fragmentation increases with increasing this parameter. The main fragmentation steps seem to involve the cleavage of the SiN bond and the conversion of the –NMe2 group.magnified image

Towards de novo identification of metabolites by analyzing tandem mass spectra

Mass spectrometry is among the most widely used technologies in proteomics and metabolomics. Being a high-throughput method, it produces large amounts of data that necessitates an automated analysis of the spectra. Clearly, database search methods for protein analysis can easily be adopted to analyze metabolite mass spectra. But for metabolites, de novo interpretation of spectra is even more important than for protein data, because metabolite spectra databases cover only a small fraction of naturally occurring metabolites: even the model plant Arabidopsis thaliana has a large number of enzymes whose substrates and products remain unknown. The field of bio-prospection searches biologically diverse areas for metabolites which might serve as pharmaceuticals. De novo identification of metabolite mass spectra requires new concepts and methods since, unlike proteins, metabolites possess a non-linear molecular structure. In this work, we introduce a method for fully automated de novo identification of metabolites from tandem mass spectra. Mass spectrometry data is usually assumed to be insufficient for identification of molecular structures, so we want to estimate the molecular formula of the unknown metabolite, a crucial step for its identification. The method first calculates all molecular formulas that explain the parent peak mass. Then, a graph is build where vertices correspond to molecular formulas of all peaks in the fragmentation mass spectra, whereas edges correspond to hypothetical fragmentation steps. Our algorithm afterwards calculates the maximum scoring subtree of this graph: each peak in the spectra must be scored at most once, so the subtree shall contain only one explanation per peak. Unfortunately, finding this subtree is NP-hard. We suggest three exact algorithms (including one fixed parameter tractable algorithm) as well as two heuristics to solve the problem. Tests on real mass spectra show that the FPT algorithm and the heuristics solve the problem suitably fast and provide excellent results: for all 32 test compounds the correct solution was among the top five suggestions, for 26 compounds the first suggestion of the exact algorithm was correct. http://www.bio.inf.uni-jena.de/tandemms

Nanoparticle MALDI-TOF Mass Spectrometry without Fragmentation: Au25(SCH2CH2Ph)18 and Mixed Monolayer Au25(SCH2CH2Ph)18−x(L)x

Intact molecular ions of the organothiolate-protected nanoparticle Au25(SCH2CH2Ph)18, including their isotopic resolution, can be observed at 7391 Da as 1- and 1+ ions in negative and positive mode, respectively, by MALDI-TOF mass spectrometry when using a tactic of threshold laser pulse intensities and trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) as matrix. Previous MALDI-TOF studies of Au nanoparticles using other matrices have encountered extensive fragmentation of nanoparticle as well as thiolate ligands. Absence of fragmentation enables precise determination of the distribution of mixed monolayer compositions on nanoparticles prepared by ligand exchange reactions and by synthesis using thiol mixtures. Reaction conditions producing mixed monolayers containing only one or a small number of usefully functional ligands can be readily identified. At increased laser pulse intensity, the first fragmentation step(s) for the Au25(SCH 2CH2Ph)18 nanoparticle results in losses of AuL units and, in particular, loss of Au4(SCH2CH2Ph)4.

Synthesis of a nanocrystalline composite W–25 wt.%Ag powder by high energy milling

W–Ag pseudo-alloys are used in electric contacts of circuit breakers. They are produced by sintering of W–Ag powder mixtures followed by hot pressing or rolling to increase density. High energy milling (HEM) can enhance sintering of systems that present low sinterability because it improves dispersion, promotes refinement of the phases and produces composite particles. This work investigates the effect of HEM upon a W–25 wt.%Ag powder mixture. Phase dispersion, evolution of the shape and size of the particles during milling and the influence of strain on the crystal structure are investigated. The milled powders consist of composite particles that were formed in the first 25 h of milling. Longer milling times improve the distribution of phases inside the composite particles. The formation of the composite particles involves sequential steps of deformation, fragmentation, cold welding, work hardening and piercing of particles of the hard phase in the soft phase. The crystal lattice of W and Cu is damaged, but is not amorphized.

LC–high-resolution multiple stage spectrometric analysis of diuretic compounds: Unusual mass fragmentation pathways

The analysis of diuretic compounds has become of great concern because of their extensive use both in therapy and in illicit treatments (such as masking agents in sport doping and drug abuse). The variety of chemical structures of this class of drugs encouraged the development of new methods and techniques of analysis, especially as regards to acidic compounds. LC/MS has so grown to be the reference technique for this kind of analysis in forensic and anti-doping confirmation purposes. Multiple stage MS permits identification of single drugs with high selectivity, but some unexpected pathways could weaken the entire process. In this work we aim to explain some unusual fragmentation steps using high-resolution MS n . For example, in the case of amiloride an intense product ion in MS 3 analysis generates an apparent loss of 10 Da. Water adduct formation and successive carbon monoxide elimination can explain this uncommon behavior, which was studied using different ion traps. Bendroflumethiazide MS n spectra show instead three successive HF losses, in spite of the presence of a radical site in the parent structure. Homolytic cleavages with radical ion production occur also in the case of protonated positive ion of ethacrynic acid (loss of chlorine radical) showing that such fragmentation behavior is not so rare as generally reported. Different ionization modes were studied and a tentative correlation with acidic-base properties was done. Multiple stage high-resolution mass spectra of positive and negative ions were discussed.

A computational protocol to fragment-based drug design at PDB scale

Fragment- based drug design has emerged in the last decade. This exciting field has been recently reviewed [1]. Obtaining structural information on the fragment complexed to the protein target is a key factor and also a major limitation. Therefore, computational methods are needed to mine efficiently all the available 3D structures of ligands complexed to proteins, both treated as a whole and as smaller fragment to increase the likelihood of fragment hopping from one target to another. MED-SuMo [2] is a target based drug design tool. This tool is based on the identification of local shape and 3D Surface Chemical Features similarities in the target binding site with other proteins (with their co-crystallized ligands). MED-SuMo uses the binding site of the target as a query to search either the whole PDB (or any corporate protein structure databank) for all the binding sites that display a local match with the query. This valuable information can then be used to identify which residues of the binding site are potentially important for affinity and selectivity. As the similar binding sites are overlaid, the co-crystallized ligands are aligned and are therefore a starting material for ligand hybridization. Among the hundreds of overlaid binding sites generated by MED-SuMo, we found the protein-ligand complexes overlaid by Pierce et al. [3] for ligand hybridization in the BREED method. Interestingly, they found relevant combinations of fragments starting from only a few protein-ligand complexes structures and we believe that the output of MED-SuMo is a very promising input for automatic methods like BREED. In this work, we used MED-SuMo on a fragment database derived from the PDB: each pdb file is converted into one or more pdb files containing a single fragment as ligand. MED-SuMo is used to query and mine the Surface Chemical Functions surrounding the fragments, seeking similarities with the protein of interest. A library of fragments positioned in the active site and annotated with the counts of contacts and h-bonds is generated. This is the basis of a protocol which ranks and hybridizes fragments. Fragmentation and hybridization steps are coupled with substructure searches in a database of chemical compounds from suppliers to increase the likelihood of fragments and hybridized molecules as synthetically feasible molecules.

Confirmation of Fenthion Metabolites in Oranges by IT-MS and QqTOF-MS

Identification of degradation products of the organophophorous pesticide fenthion formed in two orange varieties, Valencia Navel and Navel Late, under field conditions has been assessed using liquid chromatography quadrupole time-of-flight mass spectrometry and ion trap mass spectrometry. The structural elucidation of the metabolites was accomplished by the accurate mass measurements provided by the quadrupole time-of-flight mass spectrometer in MS and MS/MS modes. This instrument achieved elemental composition diagnosis for the precursor and product ions with absolute mass error of <5 ppm, which unambiguously establishes the identity of the metabolites even at low concentration. The presence of these compounds was also confirmed by electrospray ionization-ion trap mass spectrometry, performing successive fragmentation steps (MS(n)). Once identified, each molecule was confirmed by comparison with its analytical standard, also used to explore the quantitative capabilities of both mass analyzers. The extraction method was evaluated because it predetermines the metabolites that can be found (e.g., according to their polarity). Recoveries ranged from 70% for fenoxon sulfoxide (the most polar) to 101% for fenthion (the most apolar), which also indicates the method's facility to extract other more polar metabolites if present. Satisfactory linear range (r > 0.99) of more than 2 orders of magnitude was obtained with both analyzers for standards prepared in methanol and in untreated orange extracts. However, the matrix-matched standards showed suppression of the mass signal due to the matrix effect, especially for fenoxon sulfoxide and sulfone. The limits of quantification ranged from 0.005 to 0.015 mg/kg. The QqTOF-MS provided better quantification limits for fenthion and its sulfoxide and sulfone than the IT-MS. The resulting fenthion degration curves in oranges indicated that it was mainly degraded by sunlight photolysis to its sulfoxide and sulfone. However, hydrolysis was also observed by the appearance of fenoxon, fenoxon sulfoxide, and fenoxon sulfone, but always in low concentrations, which can be related to the rain events.

Comprehensive characterization of the photodissociation pathways of protonated tryptophan

The photofragmentation of protonated tryptophan has been investigated in a unique experimental setup, in which ion and neutral issued from the photofragmentation are detected in coincidence, in time and in position. From these data are extracted the kinetic energy, the number of neutral fragments associated with an ion, their masses, and the order of the fragmentation steps. Moreover, the fragmentation time scale ranging from tens of nanoseconds to milliseconds is obtained. From all these data, a comprehensive fragmentation mechanism is proposed.

Optimization of an improved analytical method for the determination of 1-nitropyrene in milligram diesel soot samples by high-performance liquid chromatography–mass spectrometry

A method for determination of nitrated polycyclic aromatic hydrocarbons (NPAHs) in diesel soot by high-performance liquid chromatography–mass spectrometry with atmospheric pressure chemical ionization (APCI) and detection by ion-trap following ultrasonic extraction is described. The determination of 1-nitropyrene that it is the predominant NPAH in diesel soot was emphasized. Vaporization and drying temperatures of the APCI interface, electronic parameters of the MS detector and the analytical conditions in reversed-phase HPLC were optimized. The patterns of fragmentation of representative NPAHs were evaluated by single and multiple fragmentation steps and negative ionization led to the largest signals. The transition (247 → 217) was employed for quantitative analysis of 1-nitropyrene. Calibration curves were linear between 1 and 15 μg L −1 with correlation coefficients better than 0.999. Typical detection limit (DL) of 0.2 μg L −1 was obtained. Samples of diesel soot and of the reference material (SRM-2975, NIST, USA) were extracted with methylene chloride. Recoveries were estimated by analysis of SRM 2975 and were between 82 and 105%. DL for 1-nitropyrene was better than 1.5 mg kg −1, but the inclusion of an evaporation step in the sample processing procedure lowered the DL. The application of the method to diesel soot samples from bench motors showed levels ≤1 μg of 1-nitropyrene per gram of diesel soot.

Ring Closing Metathesis/Fragmentation Route to (Z)‐Configured Medium Ring Cycloalkenes. Total Synthesis of (.+‐.)‐Periplanone C.

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