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At least 793 records · Page 44Linked to original sources
PubMed2005

FINDMOL: automated identification of macromolecules in electron-density maps.

Automating the determination of novel macromolecular structures via X-ray crystallographic methods involves building a model into an electron-density map. Unfortunately, the conventional crystallographic asymmetric unit volumes are usually not well matched to the biological molecular units. In most cases, the facets of the asymmetric unit cut the molecules into a number of disconnected fragments, rendering interpretation by the crystallographer significantly more difficult. The FINDMOL algorithm is designed to quickly parse the arrangement of trace points (pseudo-atoms) derived from a skeletonized electron-density map without requiring higher level prior information such as sequence information or number of molecules in the asymmetric unit. The algorithm was tested with a variety of density-modified maps computed with medium- to low-resolution data. Typically, the resulting volume resembles the biological unit. In the remaining cases the number of disconnected fragments is very small. In all examples, secondary-structural elements such as alpha-helices or beta-sheets are easily identifiable in the defragmented arrangement. FINDMOL can greatly assist a crystallographer during manual model building or in cases where automatic model building can only build partial models owing to limitations of the data such as low resolution and/or poor phases.

Algorithms↗
PubMed2003

Synthesis and biological activities of turkesterone 11alpha-acyl derivatives.

Turkesterone is a phytoecdysteroid possessing an 11alpha-hydroxyl group. It is an analogue of the insect steroid hormone 20-hydroxyecdysone. Previous ecdysteroid QSAR and molecular modelling studies predicted that the cavity of the ligand binding domain of the ecdysteroid receptor would possess space in the vicinity of C-11/C-12 of the ecdysteroid. We report the regioselective synthesis of a series of turkesterone 11alpha-acyl derivatives in order to explore this possibility. The structures of the analogues have been unambiguously determined by spectroscopic means (NMR and low-resolution mass spectrometry). Purity was verified by HPLC. Biological activities have been determined in Drosophila melanogaster B(II) cell-based bioassay for ecdysteroid agonists and in an in vitro radioligand-displacement assay using bacterially-expressed D. melanogaster EcR/USP receptor proteins. The 11alpha-acyl derivatives do retain a significant amount of biological activity relative to the parent ecdysteroid. Further, although activity initially drops with the extension of the acyl chain length (C2 to C4), it then increases (C6 to C10), before decreasing again (C14 and C20). The implications of these findings for the interaction of ecdysteroids with the ecdysteroid receptor and potential applications in the generation of affinity-labelled and fluorescently-tagged ecdysteroids are discussed.

Animals↗
PubMed2003

High resolution electron spin resonance microscopy.

NMR microscopy is routinely employed in fields of science such as biology, botany, and materials science to observe magnetic parameters and transport phenomena in small scale structures. Despite extensive efforts, the resolution of this method is limited (>10 microm for short acquisition times), and thus cannot answer many key questions in these fields. We show, through theoretical prediction and initial experiments, that ESR microscopy, although much less developed, can improve upon the resolution limits of NMR, and successfully undertake the 1 mum resolution challenge. Our theoretical predictions demonstrate that existing ESR technology, along with advanced imaging probe design (resonator and gradient coils), using solutions of narrow linewidth radicals (the trityl family), should yield 64 x 64 pixels 2D images (with z slice selection) with a resolution of 1 x 1 x 10 microm at approximately 60 GHz in less than 1h of acquisition. Our initial imaging results, conducted by CW ESR at X-band, support these theoretical predictions and already improve upon the previously reported state-of-the-art for 2D ESR image resolution achieving approximately 10 x 10 mum, in just several minutes of acquisition time. We analyze how future progress, which includes improved resonators, increased frequency of measurement, and advanced pulsed techniques, should achieve the goal of micron resolution.

Electron Spin Resonance Spectroscopy↗
PubMed2005

Determination of the size and structure of an X-pinch x-ray source from the diffraction pattern produced by microfabricated slits.

X-pinch plasma emits subnanosecond bursts of x rays in the 3-10-keV energy range from a small source. As such, it has been used for high-resolution point-projection imaging of small, dense, rapidly changing plasmas as well as for submillimeter-thick biological samples. In addition to the effect of source size on geometric resolution, a small source size can also provide high spatial coherence of x rays, enabling the rays to be used for imaging weakly absorbing objects with excellent spatial resolution by a method called phase-contrast imaging. To determine the source size, we microfabricated gold slits and imaged them in a point-projection radiography configuration. The shape of the shadow image pattern depends on the source size and energy band of the x rays, the shape and material used for the slits, and the geometry of the experiment. Experimental results have been compared with wave-optics calculations of the expected image pattern as a function of all the parameters listed above. For example, assuming a Gaussiansource distribution, an effective source size in 2.5-4.1 A radiation (1 A = 0.1 nm) of 1.2 +/- 0.5 microm (full width at half-maximum) was determined for a 20-microm Mo wire X pinch. Characterization of the size and structure of the x-ray bursts from X pinches by the use of different wire materials and different slit structures is made.

Journal Article↗
PubMed2005

Dioxins and furans determination in postmortem blood by gas chromatography--high-resolution mass spectrometry.

Dioxins and related compounds (furans) are persistent environmental contaminants that cause adverse biological effects. Their influence on humans is still unclear, except for accidental high-dose exposure. However, chronic exposure to these compounds seems to be involved in cancer, endocrine disruption, and neurobehavioral effects. For several years, a large concern about the potential health risks of dioxins is emerging in Europe and United States. Dioxin levels in biological specimens are extremely low and require very sensitive and specific methods of analysis. In this study, gas chromatography coupled to high-resolution mass spectrometry was used to evaluate dioxin body burden of two women deceased from generalized cancer. Fat fraction of blood specimens was obtained after precipitation with ethanol and extraction of both liquid and solid phases spiked with labeled 13C12-dioxin analogues. Organic phases were grouped, washed, and evaporated to weigh the lipid content. Lipids were dissolved in hexane, hydrolyzed with concentrated sulfuric acid, and discarded during water washes. Dioxins purification was achieved using three successive columns: silica, alumina/sodium sulfate, and carbon/Celite. Finally, the toluene eluent was evaporated and the extract injected in the analytical system. After chromatographic separation, detection was achieved in single ion monitoring mode using a high-resolution mass spectrometer operating in electron impact ionization mode (40 eV, minimal resolution of 10,000). Dioxin levels were expressed in pg TEQ/g of fat as defined by the World Health Organization. Quantification limits for each dioxin congener ranged from 2.5 to 12.0 pg/g fat with a relative extraction recovery always higher than 60%. Dioxin concentrations in the blood of the two deceased women were 35.0 and 42.7 pg TEQ/g fat, respectively. These concentrations are largely lower than those observed after accidental releases, but in the range of those observed in the general European population. Therefore, it was not possible to correlate dioxin body burden of the two women as a potential contributor of their cancer pathology. Nevertheless, knowledge of dioxin body burden in the French population would be of interest for an accurate interpretation of these results.

Aged↗
PubMed2002

Adaptive aberration correction in a confocal microscope.

The main advantage of confocal microscopes over their conventional counterparts is their ability to optically "section" thick specimens; the thin image slices thus obtained can be used to reconstruct three-dimensional images, a capability which is particularly useful in biological applications. However, it is well known that the resolution and optical sectioning ability can be severely degraded by system or specimen-induced aberrations. The use of high aperture lenses further exacerbates the problem. Moreover, aberrations can considerably reduce the number of photons that reach the detector, leading to lower contrast. It is rather unfortunate, therefore, that in practical microscopy, aberration-free confocal imaging is rarely achieved. Adaptive optics systems, which have been used widely to correct aberrations in astronomy, offer a solution here but also present new challenges. The optical system and the source of aberrations in a confocal microscope are considerably different and require a novel approach to wavefront sensing. This method, based upon direct measurement of Zernike aberration modes, also exhibits an axial selectivity similar to that of a confocal microscope. We demonstrate an adaptive confocal fluorescence microscope incorporating this modal sensor together with a deformable membrane mirror for aberration correction. Aberration corrected images of biological specimens show considerable improvement in contrast and apparent restoration of axial resolution.

Fourier Analysis↗
PubMed1993

Diffusion-weighted MR microscopy with fast spin-echo.

A diffusion-weighted fast spin-echo (FSE) imaging sequence for high-field MR microscopy was developed and experimentally validated in a phantom and in a live rat. Pulsed diffusion gradients were executed before and after the initial 180 degrees pulse in the FSE pulse train. This produced diffusion-related reductions in image signal intensity corresponding to gradient ("b") factors between 1.80 and 1352 s/mm2. The degree of diffusion weighting was demonstrated to be independent of echo train length for experiments using trains up to 16 echoes long. Quantitative measurements on a phantom and on a live rat produced diffusion coefficients consistent with literature values. Importantly, the eight- to 16-fold increase in imaging efficiency with FSE was not accompanied by a significant loss of spatial resolution or contrast. This permits acquisition of in vivo three-dimensional data in time periods that are appropriate for evolving biological processes. The combination of accurate diffusion weighting and high spatial resolution provided by FSE makes the technique particularly useful for MR microscopy.

Animals↗
PubMed2004

[Application of atomic force microscopy in cell biology].

The atomic force microscopy(AFM), an important instrument for the study of cell biology, has been used to image the living cells and localize the cell surface receptors under physiological conditions by perfect high resolution. It was also utilized in the investigations of the cytoskeleton, biological process and interactions between cells. The applications of AFM in cell biology have been achieved greatly in recent years and many results present a good promise in biomedical and clinical medical fields.

Cell Physiological Phenomena↗
PubMed1979

Identification and quantitation of nucleosides, bases and other UV-absorbing compounds in serum, using reversed-phase high-performance liquid chromatography. I. Chromatographic methodology.

A comprehensive investigation of the high-performance liquid chromatographic separation of nucleosides, their bases and other low-molecular-weight UV-absorbing compounds that might be found in serum is reported. A buffer-methanol gradient was used in conjunction with chemically bonded, microparticulate columns to separate many of the biologically important compounds under study in minimal time with maximal resolution. Retention data, absorbance ratios (280/254 nm) and fluorescence responses are reported for 86 nucleosides, bases, nucleotides and other UV-absorbing compounds commonly encountered in biological studies.

Amino Acids↗
PubMed2006

EMatch: an efficient method for aligning atomic resolution subunits into intermediate-resolution cryo-EM maps of large macromolecular assemblies.

Structural analysis of biological machines is essential for inferring their function and mechanism. Nevertheless, owing to their large size and instability, deciphering the atomic structure of macromolecular assemblies is still considered as a challenging task that cannot keep up with the rapid advances in the protein-identification process. In contrast, structural data at lower resolution is becoming more and more available owing to recent advances in cryo-electron microscopy (cryo-EM) techniques. Once a cryo-EM map is acquired, one of the basic questions asked is what are the folds of the components in the assembly and what is their configuration. Here, a novel knowledge-based computational method, named EMatch, towards tackling this task for cryo-EM maps at 6-10 A resolution is presented. The method recognizes and locates possible atomic resolution structural homologues of protein domains in the assembly. The strengths of EMatch are demonstrated on a cryo-EM map of native GroEL at 6 A resolution.

Algorithms↗
PubMed2006

Recent results on hydrogen and hydration in biology studied by neutron macromolecular crystallography.

Neutron diffraction provides an experimental method of directly locating hydrogen atoms in proteins, a technique complimentary to ultra-high-resolution [1, 2] X-ray diffraction. Three different types of neutron diffractometers for biological macromolecules have been constructed in Japan, France and the United States, and they have been used to determine the crystal structures of proteins up to resolution limits of 1.5-2.5 A. Results relating to hydrogen positions and hydration patterns in proteins have been obtained from these studies. Examples include the geometrical details of hydrogen bonds, H/D exchange in proteins and oligonucleotides, the role of hydrogen atoms in enzymatic activity and thermostability, and the dynamical behavior of hydration structures, all of which have been extracted from these structural results and reviewed. Other techniques, such as the growth of large single crystals, the preparation of fully deuterated proteins, the use of cryogenic techniques, and a data base of hydrogen and hydration in proteins, will be described.

Biology↗
PubMed2001

X-ray analysis of an RNA tetraplex (UGGGGU)(4) with divalent Sr(2+) ions at subatomic resolution (0.61 A).

Four-stranded guanine tetraplexes in RNA have been identified to be involved in crucial biological functions, such as dimerization of retroviral RNA, translational repression, and mRNA turnover. However, the structural basis for these biological processes is still largely unknown. Here we report the RNA tetraplex structure (UGGGGU)(4) at ultra-high resolution (0.61 A). The space group is P42(1)2, and cell constants are a = b = 36.16 A and c = 74.09 A. The structure was solved by the multiple-wavelength anomalous dispersion method using a set of three-wavelength data of the isomorphous bromo derivative (br)UGGGGU and refined to 0.61-A resolution. Each of the four strands in the asymmetric unit forms a parallel tetraplex with symmetry-related molecules. The tetraplex molecules stack on one another in opposite polarity (head-to-head or tail-to-tail) to form a pseudocontinuous column. All of the 5'-end uridines rotate around the backbone of G2, swing out, and form unique octaplexes with the neighboring G tetraplexes, whereas the 3'-end uridines are stacked-in and form uridine tetrads. All of the bases are anti, and the riboses are in the mixed C2'- and C3'-puckering mode. Strontium ions are observed in every other guanine tetrad plane, sitting on the fourfold axis and associated to the eight O6 atoms of neighboring guanine bases in a bipyramidal-antiprism geometry. The hydrogens are clearly observed in the structure.

Base Pairing↗
PubMed1986

Synthesis and biological activity of resolved C-10 diastereomers of 10-methyl- and 10-ethyl-10-deazaminopterin.

Synthesis and evaluation of the antitumor drugs 10-methyl- and 10-ethyl-10-deazaminopterin (15a,b) were previously reported for the diastereomeric mixtures, lacking resolution at the C-10 position. In order to assess biological properties of the individual diastereomers, the C-10 isomers of 4-amino-4-deoxy-10-methyl- and 10-ethyl-10-deazapteroic acids (13a,b) were prepared by total synthesis. Coupling with L-glutamate afforded the appropriate diastereomers of the title compounds. Biochemical, transport, and cell growth inhibitory properties in L1210 cells and folate-dependent bacteria were measured. Differences were generally less than 2-fold between diastereomeric pairs, but a factor of 3 was noted for d,L-15b vs. l,L-15b in inhibition of DHFR from L1210 cells and in cytotoxicity toward L1210 cells. An in vivo comparison of the isomers of 15b with racemic compound against L1210 in mice did not show a significant efficacy difference (ILS) among the compounds. However, d,L-15b showed an acute toxicity about 2.5 times that of l,L-15b.

Aminopterin↗
PubMed1992

Three-dimensional NMR microscopy: improving SNR with temperature and microcoils.

It is widely held that the spatial resolution achievable by NMR microscopic imaging is limited in biological systems by diffusion to approximately 1-5 microns. However, these estimates were developed for specific imaging techniques and represent practical rather than fundamental limits. NMR imaging is limited by the signal-to-noise ratio (SNR). Diffusion effects on spatial resolution can be made arbitrarily small in principle by increasing the gradient strength. The exponential signal attenuation from random spin motion in a gradient, however, will reduce the signal far below the noise level when the voxel size is reduced much below 5 microns. Two factors can be optimized to improve the SNR: (1) the inductive linkage between microscopic samples and the detection apparatus and (2) the temperature of the rf probe. In this work, the filling factor was optimized using inductors with diameters less than 1 mm. It is furthermore shown that probe circuit cooling results in significant improvements in SNR, whereas cooling of the preamplifier is of little value when proper noise matching between the resonant circuit and preamplifier is accomplished. Using three-dimensional Fourier imaging techniques, we have obtained images of single-cell organisms with spatial resolution of approximately 6 microns. Practical limitations include mechanical stability of the apparatus, thermal shielding between the sample and probe, and the magnetic susceptibility of the sample.

Chlorophyta↗
PubMed2000

Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution.

Halorhodopsin, an archaeal rhodopsin ubiquitous in Haloarchaea, uses light energy to pump chloride through biological membranes. Halorhodopsin crystals were grown in a cubic lipidic phase, which allowed the x-ray structure determination of this anion pump at 1.8 angstrom resolution. Halorhodopsin assembles to trimers around a central patch consisting of palmitic acid. Next to the protonated Schiff base between Lys(242) and the isomerizable retinal chromophore, a single chloride ion occupies the transport site. Energetic calculations on chloride binding reveal a combination of ion-ion and ion-dipole interactions for stabilizing the anion 18 angstroms below the membrane surface. Ion dragging across the protonated Schiff base explains why chloride and proton translocation modes are mechanistically equivalent in archaeal rhodopsins.

Bacteriorhodopsins↗
PubMed1997

Greedy Algorithms for Finding a Small Set of Primers Satisfying Cover and Length Resolution Conditions in PCR Experiments.

Selecting a good collection of primers is very important for polymerase chain reaction (PCR) experiments. Most existing algorithms for primer selection are concerned with computing a primer pair for each DNA sequence. In generalizing the arbitrarily primed PCR, etc., to the case that all DNA sequences of target objects are already known, like about 6000 ORFs of yeast, we may design a small set of primers so that all the targets are PCR amplified and resolved electrophoretically in a series of experiments. This is quite useful because deceasing the number of primers greatly reduces the cost of experiments. Pearson et al. (ISMB 1995: 285-291, 1995; Discrete Appl. Math. 71: 231-246, 1996) consider finding a minimum set of primers covering all given DNA sequences, but their method does not meet necessary biological conditions such as primer amplification and electrophoresis resolution. In this paper, based on the modeling and computational complexity analysis by Doi, we propose algorithms for this primer selection problem. These algorithms do not necessarily minimize the number of primers, but, since basic versions of these problems are shown to be computationally intractable, especially even for approximability with the length resolution condition, this is inevitable. In the algorithms, the amplification condition by a primer pair and the length resolution condition by electrophoresis are incorporated. These algorithms are based on the theoretically well-founded greedy algorithm for the set cover in computer science. Preliminary computational results are presented to show the validity of this approach. The number of computed primers is much less than a half of the number of targets, and hence is less than one forth of the number needed in the multiplex PCR.

Journal Article↗
PubMed1989

High resolution scanning electron microscopy of the cell.

The scanning electron microscope (SEM) has become a powerful tool for ultrastructural research with improvement of the instrument's resolution and progress in specimen preparation techniques. With regard to resolution, it has been improved step-by-step in this decade and, in 1985, an ultra-high resolution SEM (UHS-T1) was developed, with a resolution of 0.5 nm. Concerning specimen preparation, the osmium-DMSO-osmium method, which is effective for revealing intracellular structures, has come to be widely used. Techniques for observing smaller objects, such as bacteriophages, viruses, and biological macromolecules, have also been devised in recent years. As a result of these preparation techniques and the availability of the ultra-high resolution SEM, the application of SEM in biology is expanding rapidly. In this paper, an outline of the ultra-high resolution SEM, techniques for specimen preparation, findings of some biological materials by these techniques, and guidelines to making the specimens, are described.

Animals↗