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Biomedical subjects

Andrew N Lane

Publications and source records attributed to Andrew N Lane.

9 recordsLinked to original sources
PubMed2006

Integrating metabolomics and transcriptomics for probing SE anticancer mechanisms.

Transcriptomics provides the tool for deciphering gene expression networks, and proteomics links these networks to protein products. The third crucial partner is metabolomics, which defines the metabolic network(s) linked to gene expression. NMR and mass spectrometry enable the broad screen analysis of the metabolome and its transformation pathways, transcending classical targeted metabolic studies. These tools were combined to investigate the anticancer mechanisms of different selenium forms in human lung cancer cells. Using 2-D NMR and tandem-MS, we mapped perturbations of 13C labeling patterns in numerous metabolites induced by selenite and selenomethionine. This information was used to interpret selenite-induced changes in gene expression networks. Linking metabolic dysfunctions to altered gene expression profiles provided new insights into the regulatory network underlying the metabolic dysfunctions, enabled the assembly of discrete gene expression events into functional pathways, and revealed protein targets for proteomic analysis.

Animals↗
PubMed2005

Determining the origin of the stabilization of DNA by 5-aminopropynylation of pyrimidines.

DNA duplexes are stabilized by aminopropynyl modification of pyrimidines at the 5 position. A combination of thermodynamic analyses as a function of ionic strength, NMR, and molecular modeling has been applied to determine the origin of the stabilization. UV melting studies of a dodecamer bearing one, two, or three nonadjacent modified dU and dC and of a single dU(8) in the Dickerson-Drew dodecamer revealed that the modifications are essentially additive in terms of T(m), DeltaG, and DeltaH, and there is little difference between dU and dC. The free energy change was parsed into electrostatic and nonelectrostatic components, which showed a significant contribution from charge interactions at physiological ionic strength but also a nonelectrostatic contribution that arises in part from hydration. NMR spectroscopy of the modified Dickerson-Drew dodecamer revealed that the conformation of the duplexes is not significantly altered by the modifications, though (31)P NMR shows that the positive charge may affect ionic interactions with the oxygen atoms of the neighboring phosphates. The modified duplex showed significant hydration in both major and minor grooves. The single strands were also analyzed by NMR, which showed evidence of significant stacking interactions in the modified oligonucleotide. Parsing the energy contribution has shown that electrostatics and hydration can produce substantial increases in thermodynamic stability without significant changes in the conformation of the duplex state. These considerations have significance for the design of oligonucleotides used for hybridization.

Alkynes↗
PubMed2005

Improving NMR sensitivity in room temperature and cooled probes with dipolar ions.

The response of inverse triple resonance cold and conventional probes to ionic strength has been compared under a variety of conditions relevant to protein NMR. Increasing the salt concentration degrades probe performance in terms of sensitivity, and the effect is more severe for cold probes and with increasing magnetic field strength. This is especially noticeable for experiments that involve a spin lock or decoupling, where sensitivity losses compared with pure water can be more than 2-fold. We have investigated the use of glycine as a substitute for salt as a supporting solute for proteins, and we show that it has a minimal effect on probe tuning or performance. Readily available d5-Gly is a useful co-solute for protein NMR, especially at high magnetic field strengths and on cold probes, as it maintains solubility while not degrading probe performance.

Animals↗
PubMed2004

Secondary structure and stability of the selenocysteine insertion sequences (SECIS) for human thioredoxin reductase and glutathione peroxidase.

We have used high resolution NMR and thermodynamics to characterize the secondary structure and stability of the selenocysteine insertion sequences (SECIS) of human glutathione peroxidase (58 nt) and thioredoxin reductase (51 nt). These sequences are members of the two classes of SECIS recently identified with two distinct structures capable of directing selenocysteine incorporation into proteins in eukaryotes. UV melting experiments showed a single cooperative and reversible transition for each RNA, which indicates the presence of stable secondary structures. Despite their large size, the RNAs gave well resolved NMR spectra for the exchangeable protons. Using NOESY, the imino protons as well as the cytosine amino protons of all of the Watson-Crick base pairs were assigned. In addition, a number of non-canonical base pairs including the wobble G.U pairs were identified. The interbase-pair NOEs allowed definition of the hydrogen-bonded structure of the oligonucleotides, providing an experimental model of the secondary structure of these elements. The derived secondary structures are consistent with several features of the predicted models, but with some important differences, especially regarding the conserved sequence motifs.

Base Sequence↗
PubMed2004

The promise of metabolomics in cancer molecular therapeutics.

A systematic elucidation of cancer cell dysfunction and therapeutic mechanisms seems within reach with modern functional genomics and proteomics tools. However, for this potential to be realized, the metabolic consequences of gene expression and protein activity must be understood. 'Metabolomics' is currently a major missing component. It differs from classical metabolic studies by its greater breadth, depth and speed, enabled by the huge advances in analytical instrumentation (especially nuclear magnetic resonance and mass spectrometry) over the past two decades. Multiple metabolic pathways and networks can now be traced by the flow of atoms through metabolites, known as isotopomer analyses. Thus, metabolomics demands both high-throughput and high-information content analyses, and interpretation: the latter is currently a bottleneck. There are currently very few metabolomic studies in cancer therapeutics, despite this great need and potential.

Animals↗
PubMed2003

The solution structure of a DNA*RNA duplex containing 5-propynyl U and C; comparison with 5-Me modifications.

The addition of the propynyl group at the 5 position of pyrimidine nucleotides is highly stabilising. We have determined the thermodynamic stability of the DNA.RNA hybrid r(GAAGAGAAGC)*d(GC(p)U(p)U(p)C(p)U(p) C(p)U(p)U(p)C) where p is the propynyl group at the 5 position and compared it with that of the unmodified duplex and the effects of methyl substitutions. The incorporation of the propyne group at the 5 position gives rise to a very large stabilisation of the hybrid duplex compared with the analogous 5-Me modification. The duplexes have been characterised by gel electrophoresis and NMR spectroscopy, which indicate that methyl substitutions have a smaller influence on local and global conformation than the propynyl groups. The increased NMR spectral dispersion of the propyne-modified duplex allowed a larger number of experimental restraints to be measured. Restrained molecular dynamics in a fully solvated system showed that the propyne modification leads to substantial conformational rearrangements stabilising a more A-like structure. The propynyl groups occupy a large part of the major groove and make favourable van der Waals interactions with their nearest neighbours and the atoms of the rings. This enhanced overlap may account at least in part for the increased thermodynamic stability. Furthermore, the simulations show a spine of hydration in the major groove as well as in the minor groove involving the RNA hydroxyl groups.

DNA↗
PubMed2003

NMR structure of the DNA-binding domain of the cell cycle protein Mbp1 from Saccharomyces cerevisiae.

The three-dimensional solution structure of the DNA-binding domain of Mlu-1 box binding protein (Mbp1) has been determined by multidimensional NMR spectroscopy. Mbp1 is a cell cycle transcription factor from Saccharomyces cerevisiae and consists of an N-terminal DNA-binding domain, a series of ankyrin repeats, and a heterodimerization domain at the C-terminus. A set of conformers comprising 19 refined structures was calculated via a molecular dynamics simulated annealing protocol using distance, dihedral angle, and residual dipolar coupling restraints derived from either double or triple resonance NMR experiments. The solution structure consists of a six-stranded beta-sheet segment folded against two pairs of alpha-helices in the topology of the winged helix-turn-helix family of proteins and is in agreement with the X-ray structures. In addition, the solution structure shows that the C-terminal tail region of this domain folds back and makes specific interactions with the N-terminal beta-strand of the protein. This C-terminal region is essential for full DNA-binding activity but appears in the X-ray structure to be disordered. The fold-back structure extends the region of positive electrostatic potential, and this may enhance the nonspecific contribution to binding by favorable electrostatic interactions with the DNA backbone.

Amino Acid Sequence↗
PubMedDate not supplied

An electrophoretic profiling method for thiol-rich phytochelatins and metallothioneins.

Thiol-rich peptides such as phytochelatins (PCs) and metallothioneins (MTs) are important cellular chelating agents which function in metal detoxification and/or homeostasis. The variations in molecular sizes and lack of chromophores of these peptides make their analysis difficult. This paper reports an electrophoresis-based method for a broad screen of thiol-rich peptides and proteins. The method uses the thiol-selective fluorescent tag, monobromobimane, coupled with Tricine--sodium dodecyl sulphate--urea polyacrylamide gel electrophoresis for a sensitive determination of both PCs and MTs. Results for PCs were confirmed by two-dimensional NMR and HPLC-tandem MS analyses. Sample throughput is substantially improved over chromatography-based methods through parallel sample analysis in 1 h of electrophoretic separation. The method is versatile in that peptides ranging from glutathione to large proteins can be analysed by simple modification(s) of the extraction and electrophoretic conditions, and the nature of the method supports serendipitous detection of unexpected or novel thiol metabolites.

Bridged Bicyclo Compounds↗