LDL particle size and composition and incident cardiovascular disease in a South-European population: The Hortega-Liposcale Follow-up Study.

BACKGROUND: The association of low-density lipoprotein (LDL) particle composition with cardiovascular risk has not been explored before. The aim was to evaluate the relationship between baseline LDL particle size and composition (proportions of large, medium and small LDL particles over their sum expressed as small-LDL %, medium-LDL % and large-LDL %) and incident cardiovascular disease in a population-based study. METHODS: Direct measurement of LDL particles was performed using a two-dimensional NMR-technique (Liposcale®). LDL cholesterol was assessed using both standard photometrical methods and the Liposcale® technique in a representative sample of 1162 adult men and women from Spain. RESULTS: The geometric mean of total LDL particle concentration in the study sample was 827.2 mg/dL (95% CI 814.7, 839.8). During a mean follow-up of 12.4 ±â€¯3.3 years, a total of 159 events occurred. Medium LDL particles were positively associated with all cardiovascular disease, coronary heart disease (CHD) and stroke after adjustment for traditional risk factors and treatment. Regarding LDL particle composition, the multivariable adjusted hazard ratios for CHD for a 5% increase in medium and small LDL % by a corresponding decrease of large LDL % were 1.93 (1.55, 2.39) and 1.41 (1.14, 1.74), respectively. CONCLUSIONS: Medium LDL particles were associated with incident cardiovascular disease. LDL particles showed the strongest association with cardiovascular events when the particle composition, rather than the total concentration, was investigated. A change in baseline composition of LDL particles from large to medium and small LDL particles was associated with an increased cardiovascular risk, especially for CHD.

Comparative metabolic profiling of paediatric ependymoma, medulloblastoma and pilocytic astrocytoma.

Brain tumours are the most common solid tumours in children and a major cause of childhood mortality. The most common paediatric brain tumours include ependymomas, cerebellar astrocytomas and medulloblastomas. These brain tumours are highly heterogeneous regarding their histology, prognosis and therapeutic response. Subtle biochemical changes can be detected in intact tissues by High-Resolution Proton Magnetic Angle Spinning Spectroscopy (HR-MAS) revealing the status of tumour microheterogeneity and metabolic alterations before they are morphologically detectable. In this study, we present metabolic profiles by HR-MAS of 20 intact tissue samples from paediatric brain tumours. Tumour types include ependymoma, medulloblastoma and pilocytic astrocytoma. The metabolic characterization of paediatric brain tumour tissue by HR-MAS spectroscopy provided differential patterns for these tumours. The metabolic composition of the tumour tissue was highly consistent with previous in vivo and ex vivo studies. Some resonances detected in this work and not previously observed by in vivo spectroscopy also show potential in determining tumour type and grade (fatty acids, phenylalanine, glutamate). Overall, this work suggests that the additional information obtained by NMR metabolic profiling applied to tissue from paediatric brain tumours may be useful for assessing tumour grade and determining optimum treatment strategies.

MRS as endogenous molecular imaging for brain and prostate tumors: FP6 project "eTUMOR".

Molecular imaging has become during the last years in an important tool for supporting cancer diagnosis and prognosis. PET and SPECT are the most common molecular imaging techniques, although very promising and specific biological molecular agent contrast for CT and MRI are being recently developed. However, the above imaging techniques require exogenous contrast agents and usually a sole molecular image can be obtained at once. On the contrary, in vivo magnetic resonance spectroscopy (MRS), in particular 1H MRS can simultaneously provide several molecular images using endogenous metabolites. In addition to biochemical spatial information from molecular imaging spectroscopy, MRS can also provide average metabolite profile of the selected affected tissue region. Initially MRS, especially 1H MRS, was extensively applied to complete and improve the diagnosis and prognosis of central nervous system (CNS) pathologies, in particular brain tumors. However, during the last years the MRS applications have been extent to the diagnosis of different very common cancer types such as breast, prostate, colon carcinoma, and ovarian, among others. Likewise, MRS has been also used for lymph node assessment. In this contribution, the added value of MRS for the diagnosis, prognosis, and treatment selection of two different, important types of cancer: (1) brain tumors and (2) prostate, will be presented and discussed. Brain tumors are the leading cause of death in children under 15, and although in adults, brain cancers are proportionately less common than other cancers, it is a devastating disease with high mortality. There is a great need to increase our understanding of brain tumor biology to improve diagnosis and to develop new treatments. 1H MRS is currently the only noninvasive method that can be used to investigate molecular profile of brain tumors and also provide molecular images, more than six in one acquisition, of the distribution of chemicals in a tumor, which are also generally heterogeneous. A summary of the applications of 1H MRS to the in vivo diagnosis and prognosis of brain tumors will be presented. In addition, examples of metabolite limits, infiltration and high cellularity location for neurosurgery applications by MRS molecular images will be shown. Likewise, new ex vivo methods of studying the detailed biochemistry of tumor biopsies as metabolomic (high resolution magic angle spinning [HR-MAS]) and transcriptomic (DNA microarrays) will be discussed as complementary to in vivo MRS (FP6 European project eTUMOR). A preliminary comparison between molecular images from PET and 1H MRS will be also presented. Finally, the application of 1H MRS to the improvement of prostate diagnosis and prognosis, the second leading cause of cancer death, will also discussed, with particular attention to the location cancer contribution from MRS molecular images.

Solution NMR structure and folding dynamics of the N terminus of a rat non-muscle alpha-tropomyosin in an engineered chimeric protein.

Tropomyosin is an alpha-helical coiled-coil protein that aligns head-to-tail along the length of the actin filament and regulates its function. The solution structure of the functionally important N terminus of a short 247-residue non-muscle tropomyosin was determined in an engineered chimeric protein, GlyTM1bZip, consisting of the first 19 residues of rat short alpha-tropomyosin and the last 18 residues of the GCN4 leucine zipper. A gene encoding GlyTM1bZip was synthesized, cloned and expressed in Escherichia coli. Triple resonance NMR spectra were analyzed with the program AutoAssign to assign its backbone resonances. Multidimensional nuclear Overhauser effect spectra, X-filtered spectra and (3)J(H(N)-H(alpha)) scalar coupling were analyzed using AutoStructure. This is the first application of this new program to determine the three-dimensional structure of a symmetric homodimer and a structure not previously reported. Residues 7-35 in GlyTM1bZip form a coiled coil, but neither end is helical. Heteronuclear (15)N-(1)H nuclear Overhauser effect data showed that the non-helical N-terminal residues are flexible. The (13)C' chemical shifts of the coiled-coil backbone carbonyl groups in GlyTM1bZip showed a previously unreported periodicity, where resonances arising from residues at the coiled-coil interface in a and d positions of the heptad repeat were displaced relatively upfield and those arising from residues in c positions were displaced relatively downfield. Heteronuclear single quantum coherence spectra, collected as a function of temperature, showed that cross-peaks arising from the alpha-helical backbone and side-chains at the coiled-coil interface broadened or shifted with T(M) values approximately 20 degrees C lower than the loss of alpha-helix measured by circular dichroism, suggesting the presence of a folding intermediate. The side-chain of Ile14, a residue essential for binding interactions, exhibited multiple conformations. The conformational flexibility of the N termini of short tropomyosins may be important for their binding specificity.

HYPER: a hierarchical algorithm for automatic determination of protein dihedral-angle constraints and stereospecific C beta H2 resonance assignments from NMR data.

A new computer program, HYPER, has been developed for automated analysis of protein dihedral angle values and C beta H2 stereospecific assignments from NMR data. HYPER uses a hierarchical grid-search algorithm to determine allowed values of phi, psi, and chi 1 dihedral angles and C beta H2 stereospecific assignments based on a set of NMR-derived distance and/or scalar-coupling constraints. Dihedral-angle constraints are valuable for restricting conformational space and improving convergence in three-dimensional structure calculations. HYPER computes the set of phi, psi, and chi 1 dihedral angles and C beta H2 stereospecific assignments that are consistent with up to nine intraresidue and sequential distance bounds, two pairs of relative distance bounds, thirteen homo- and heteronuclear scalar coupling bounds, and two pairs of relative scalar coupling constant bounds. The program is designed to be very flexible, and provides for simple user modification of Karplus equations and standard polypeptide geometries, allowing it to accommodate recent and future improved calibrations of Karplus curves. The C code has been optimized to execute rapidly (0.3-1.5 CPU-sec residue-1 using a 5 degrees grid) on Silicon Graphics R8000, R10000 and Intel Pentium CPUs, making it useful for interactive evaluation of inconsistent experimental constraints. The HYPER program has been tested for internal consistency and reliability using both simulated and real protein NMR data sets.

Homology modeling using simulated annealing of restrained molecular dynamics and conformational search calculations with CONGEN: application in predicting the three-dimensional structure of murine homeodomain Msx-1.

We have developed an automatic approach for homology modeling using restrained molecular dynamics and simulated annealing procedures, together with conformational search algorithms available in the molecular mechanics program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137-168). The accuracy of the method is validated by "predicting" structures of two homeodomain proteins with known three-dimensional structures, and then applied to predict the three-dimensional structure of the homeodomain of the murine Msx-1 transcription factor. Regions of the unknown protein structure that are highly homologous to the known template structure are constrained by "homology distance constraints," whereas the conformations of nonhomologous regions of the unknown protein are defined only by the potential energy function. A full energy function (excluding explicit solvent) is employed to ensure that the calculated structures have good conformational energies and are physically reasonable. As in NMR structure determinations, information on the consistency of the structure prediction is obtained by superposition of the resulting family of protein structures. In this paper, our homology modeling algorithm is described and compared with related homology modeling methods using spatial constraints derived from the structures of homologous proteins. The software is then used to predict the DNA-bound structures of three homeodomain proteins from the X-ray crystal structure of the engrailed homeodomain protein (Kissinger CR et al., 1990, Cell 63:579-590). The resulting backbone and side-chain conformations of the modeled yeast Mat alpha 2 and D. melanogaster Antennapedia homeodomains are excellent matches to the corresponding published X-ray crystal (Wolberger C et al., 1991, Cell 67:517-528) and NMR (Billeter M et al., 1993, J Mol Biol 234:1084-1097) structures, respectively. Examination of these structures of Msx-1 reveals a network of highly conserved surface salt bridges that are proposed to play a role in regulating protein-protein interactions of homeodomains in transcription complexes.