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At least 289 records · Page 16Linked to original sources
PubMed1979

Dark field imaging of biological macromolecules with the scanning transmission electron microscope.

A scanning transmission electron microscope (STEM) equipped with a field emission gun has been employed for the examination of biological macromolecules at high resolution. The quality of micrographs obtained with the STEM is dependent upon the quality of the substrate used to support biological objects because the image contrast in dark field is proportional to the mass density of the specimen. In order to reduce deleterious effects of the substrates on the image quality, we have developed a method of fabricating substrates consisting of very thin, very clean carbon films supported on very clean fenestrated plastic films. These films are approximately 15 A thick. Well-known biological macromolecules such as glutamine synthetase and tobacco mosaic virus (both stained) and low-density lipoprotein and ferritin (both unstained were placed on these substrates and examined with the STEM by using various modes of contrast. The micrographs obtained by using the dark field mode of contrast employing an annular detector were free from phase contrast, as expected. Using this contrast mode, we have been able to directly observe (in-focus) 2.5- to 4.4-A lattice spacings in the ferritin core. The effect of electron radiation damage on the helical structure of tobacco mosaic virus was also examined. Micrographs as well as corresponding optical diffraction patterns obtained with moderately low doses showed very clear helical structure from both sides of the virus. In addition, the (11.5 A)(-1) layer lines indicated the effective resolution attained on these particles.

Ferritins↗
PubMed2004

Electron microscopy of biological macromolecules: bridging the gap between what physics allows and what we currently can get.

The resolution achieved in low-dose electron microscopy of biological macromolecules is significantly worse than what can be obtained on the same microscopes with more robust specimens. When two-dimensional crystals are used, it is also apparent that the high-resolution image contrast is much less than what it could be if the images were perfect. Because specimen charging is one factor that might limit the contrast and resolution achieved with biological specimens, we have investigated the use of holey support films that have been coated with a metallic film before depositing specimens onto a thin carbon film that is suspended over the holes. Monolayer crystals of paraffin (C44H90) are used as a test specimen for this work because of the relative ease in imaging Bragg spacings at 0.4 nm resolution, the relative ease of measuring the contrast in these images, and the similar degree of radiation sensitivity of these crystals when compared to biological macromolecules. A metallic coating on the surrounding support film does, indeed, produce a significant improvement in the high-resolution contrast for a small fraction of the images. The majority of images show little obvious improvement, however, and even the coated area of the support film continues to show a significant amount of beam-induced movement under low-dose conditions. The fact that the contrast in the best images can be as much as 25%-35% of what it would be in a perfect image is nevertheless encouraging, demonstrating that it should be possible, in principle, to achieve the same performance for every image. Routine data collection of this quality would make it possible to determine the structure of large, macromolecular complexes without the need to grow crystals of these difficult specimen materials.

Crystallization↗
PubMed2005

Intact-protein-based high-resolution three-dimensional quantitative analysis system for proteome profiling of biological fluids.

The substantial complexity and vast dynamic range of protein abundance in biological fluids, notably serum and plasma, present a formidable challenge for comprehensive protein analysis. Integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomics analysis of biological fluids. We have implemented an orthogonal three-dimensional intact-protein analysis system (IPAS), coupled with protein tagging and immunodepletion of abundant proteins, to quantitatively profile the human plasma proteome. Following immunodepletion, plasma proteins in each of paired samples are concentrated and labeled with a different Cy dye, before mixing. Proteins are subsequently separated in three dimensions according to their charge, hydrophobicity, and molecular mass. Differences in the abundance of resolved proteins are determined based on Cy dye ratios. We have applied this strategy to profile the plasma proteome for changes that occur with acute graft-versus-host disease (GVHD), following allogeneic bone marrow transplantation (BMT). Using capillary HPLC ESI Q-TOF MS, we identified 75 proteins in the micromolar to femtomolar range that exhibited quantitative differences between the pre- and post-GVHD samples. These proteins included serum amyloid A, apolipoproteins A-I/A-IV, and complement C3 that are well-known acute-phase reactants likely reflecting the post-BMT inflammatory state. In addition, we identified some potentially interesting immunologically relevant molecules including vitamin D-binding protein, fetuin, vitronectin, proline-rich protein 3 and 4, integrin-alpha, and leukocyte antigen CD97. IPAS provides a combination of comprehensive profiling and quantitative analysis, with a substantial dynamic range, for disease-related applications.

Adult↗
PubMedDate not supplied

The Electron Microscopy Outreach Program: A Web-based resource for research and education.

We have developed a centralized World Wide Web (WWW)-based environment that serves as a resource of software tools and expertise for biological electron microscopy. A major focus is molecular electron microscopy, but the site also includes information and links on structural biology at all levels of resolution. This site serves to help integrate or link structural biology techniques in accordance with user needs. The WWW site, called the Electron Microscopy (EM) Outreach Program (URL: http://emoutreach.sdsc.edu), provides scientists with computational and educational tools for their research and edification. In particular, we have set up a centralized resource containing course notes, references, and links to image analysis and three-dimensional reconstruction software for investigators wanting to learn about EM techniques either within or outside of their fields of expertise.

Academies and Institutes↗
PubMed1989

DNA fingerprinting.

Hypervariable tandem-repetitive minisatellite regions of human DNA can be used to generate individual-specific DNA fingerprints. Validation studies have demonstrated the reliability of the analysis, the mode of inheritance of the minisatellites, and the unparalleled degree of individual specificity. The uses of hypervariable probes in forensic biology, paternity testing, and the resolution of a wide range of problems in genetics, molecular biology, population biology, and medicine are illustrated.

Animals↗
PubMed2004

Bow ties, metabolism and disease.

Highly organized, universal structures underlying biological and technological networks mediate effective trade-offs among efficiency, robustness and evolvability, with predictable fragilities that can be used to understand disease pathogenesis. The aims of this article are to describe the features of one common organizational architecture in biology, the bow tie. Large-scale organizational frameworks such as the bow tie are necessary starting points for higher-resolution modeling of complex biologic processes

Computational Biology↗
PubMed2002

Chemistry and biology in the new age.

Ahmed Zewail won the 1999 Nobel Prize in chemistry for his visionary work in probing the motions of atoms at the femtosecond level. This pioneering research, a decade earlier, opened up a new frontier of scientific knowledge. Zewail and his multidisciplinary team at Caltech are now pushing further into the realm of molecular complexity, with the ultimate aim of exploring the global dynamics of biological systems at atomic resolution. This requires a new method of 'watching' reactions--ultrafast electron diffraction.

Biology↗
PubMed2004

A novel BioXAS technique with sub-millisecond time resolution to track oxidation state and structural changes at biological metal centers.

Oxidation-state and structural changes at the metal center are crucial for the catalytic reactions of most metalloenzymes. The characterization of reaction intermediates is a prerequisite for understanding the catalytic mechanism. Frequently, intermediates are formed on the microsecond to millisecond timescale. To follow these reactions in real time represents a major challenge in structural biology. Time-resolved BioXAS is a particularly promising tool for resolving such intermediates. A novel approach for BioXAS, termed 'sampling-XAS', is presented. First room-temperature sampling-XAS results have been obtained for the manganese complex of oxygenic photosynthesis. Oxidation-state changes are monitored with a time resolution as good as 200 micros. The current prospects and limitations as well as future perspectives of time-resolved BioXAS are discussed.

Catalysis↗
PubMed1994

Specimen flatness of glucose-embedded biological materials for electron crystallography is affected significantly by the choice of carbon evaporation stock.

Imperfect specimen flatness can be a significant limitation in the application of electron crystallography to high-resolution structure analysis of biological macromolecules. We now report that the choice of solid carbon stock that is used to make evaporated carbon films can have a very great effect on the preparation of flat specimens of glucose-embedded purple membrane. The degree of purity of the carbon does not seem to be the controlling factor, and other likely factors such as the type of mica used as a substrate, the evaporation apparatus used (and its limiting vacuum), and the use of a continuous versus an interrupted evaporation protocol do not have a discernible influence. The physical or chemical basis for the observed differences in specimen flatness is still unknown; however, the important conclusion that we can communicate at this point is that the choice of evaporating material does have a major effect on the flatness of purple membrane, the specimen used here. The implication is that different sources of carbon stock should be tried whenever difficulty is encountered in the preparation of suitably flat specimens of biological macromolecules.

Crystallography↗
PubMedDate not supplied

Ultrasound-mediated biophotonic imaging: a review of acousto-optical tomography and photo-acoustic tomography.

This article reviews two types of ultrasound-mediated biophotonic imaging-acousto-optical tomography (AOT, also called ultrasound-modulated optical tomography) and photo-acoustic tomography (PAT, also called opto-acoustic or thermo-acoustic tomography)-both of which are based on non-ionizing optical and ultrasonic waves. The goal of these technologies is to combine the contrast advantage of the optical properties and the resolution advantage of ultrasound. In these two technologies, the imaging contrast is based primarily on the optical properties of biological tissues, and the imaging resolution is based primarily on the ultrasonic waves that either are provided externally or produced internally, within the biological tissues. In fact, ultrasonic mediation overcomes both the resolution disadvantage of pure optical imaging in thick tissues and the contrast and speckle disadvantages of pure ultrasonic imaging. In our discussion of AOT, the relationship between modulation depth and acoustic amplitude is clarified. Potential clinical applications of ultrasound-mediated biophotonic imaging include early cancer detection, functional imaging, and molecular imaging.

Acoustics↗
PubMed2026

3D Proteomics: Structural, Functional, Chemical and Biomarker Discovery Proteomics With LiP-MS.

Protein structural dynamics drive changes in protein function, making the capture of such dynamics essential for interrogating biological systems. Here we review limited proteolysis coupled to mass spectrometry (LiP-MS), a structural and chemical proteomics method that uses changes in susceptibility to protease cleavage to profile proteome-wide protein structural changes within complex biological samples. In the decade since its development, LiP-MS has become a broadly used structural proteomics method, with peptide-level resolution. It has identified drug targets, delineated altered cellular pathways in response to complex perturbations, revealed structural information on otherwise challenging protein targets, and demonstrated the new concept of structural biomarkers of disease. Because LiP-MS simultaneously probes numerous types of molecular events, such as molecular binding, changes in enzyme activity, chemical modifications, allosteric conformational changes, aggregation, and unfolding, it supports a new proteomics workflow which we term 3D proteomics. This workflow enables the detection of specific functional sites within proteins that are altered upon perturbation, thereby guiding the generation of molecular hypotheses. Further, by globally profiling structural in addition to protein abundance changes, LiP-MS has proven able to greatly increase the information content of functional proteomics screens. In sum, LiP-MS has supported the development of a novel conceptual framework for generating, visualizing, and interpreting structural proteomics data with peptide level resolution, thereby comprehensively probing biological systems. Here we survey the applications of LiP-MS, discuss methodological variants developed by us and others, and describe the use of this new type of omics readout for structural, functional, chemical, and biomarker discovery proteomics.

Proteomics↗
PubMed2006

Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.

Biological structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resolution. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small numbers of them at a time, at low excitation intensity. To control the number of visible fluorophores in the field of view and ensure that optically active molecules are separated by much more than the width of the point spread function, photoactivatable fluorescent molecules are used, in this case the photoactivatable green fluorescent protein (PA-GFP). For these photoactivatable molecules, the activation rate is controlled by the activation illumination intensity; nonfluorescent inactive molecules are activated by a high-frequency (405-nm) laser and are then fluorescent when excited at a lower frequency. The fluorescence is imaged by a CCD camera, and then the molecules are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The rate of photobleaching is controlled by the intensity of the laser used to excite the fluorescence, in this case an Ar+ ion laser. Because only a small number of molecules are visible at a given time, their positions can be determined precisely; with only approximately 100 detected photons per molecule, the localization precision can be as much as 10-fold better than the resolution, depending on background levels. Heterogeneities on length scales of the order of tens of nanometers are observed by FPALM of PA-GFP on glass. FPALM images are compared with images of the same molecules by widefield fluorescence. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with atomic force microscopy and show that the full width at half-maximum of features approximately 86 +/- 4 nm is significantly better than the expected diffraction-limited optical resolution. The number of fluorescent molecules and their brightness distribution have also been determined using FPALM. This new method suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.

Aluminum Oxide↗
PubMed2006

Detection and characterization of metabolites in biological matrices using mass defect filtering of liquid chromatography/high resolution mass spectrometry data.

An improved mass defect filter (MDF) method employing both drug and core structure filter templates was applied to the processing of high resolution liquid chromatography/mass spectrometry (LC/MS) data for the detection and structural characterization of oxidative metabolites with mass defects similar to or significantly different from those of the parent drugs. The effectiveness of this approach was investigated using nefazodone as a model compound, which is known to undergo multiple common and uncommon oxidative reactions. Through the selective removal of all ions that fall outside of the preset filter windows, the MDF process facilitated the detection of all 14 nefazodone metabolites presented in human liver microsomes in the MDF-filtered chromatograms. The capability of the MDF approach to remove endogenous interferences from more complex biological matrices was examined by analyzing omeprazole metabolites in human plasma. The unprocessed mass chromatogram showed no distinct indication of metabolite peaks; however, after MDF processing, the metabolite peaks were easily identified in the chromatogram. Compared with precursor ion scan and neutral loss scan techniques, the MDF approach was shown to be more effective for the detection of metabolites in a complex matrix. The comprehensive metabolite detection capability of the MDF approach, together with accurate mass determination, makes high resolution LC/MS a useful tool for the screening and identification of both common and uncommon drug metabolites.

Biotransformation↗
PubMed2003

Gel and polymer-solution mediated separation of biopolymers by capillary electrophoresis.

In the age of genomics and proteomics, high-resolution separation techniques are routinely utilized in an integrated and automated fashion to solve formidable separation problems and provide the means for large-scale analysis of biological samples with excellent resolution. By automating the current manual procedures, capillary gel (CGE) and polymer-solution mediated electrophoresis greatly enhance the productivity of biopolymer analysis while also reducing both analysis time and the human intervention necessary from sample loading to data processing. The advent of this novel and high-performance bioseparation technique has made it possible to sequence the human genome and revealed global changes in the genome and proteome level, bringing about a revolutionary transition in our views of living systems on the molecular basis. CGE and polymer-solution mediated electrophoresis and related microseparation methods (e.g., electrophoresis microchips) are quickly becoming important separation and characterization tools in analytical biochemistry and molecular biology. This review gives an overview of the key application areas of DNA, protein, and complex carbohydrate analysis, and summarizes the latest developments on CGE column technology, including capillary coatings and sieving polymer matrices. Micropreparative aspects and related microseparation techniques are also discussed.

Biopolymers↗
PubMed2003

Automatic CTF correction for single particles based upon multivariate statistical analysis of individual power spectra.

Three-dimensional electron cryomicroscopy of randomly oriented single particles is a method that is suitable for the determination of three-dimensional structures of macromolecular complexes at molecular resolution. However, the electron-microscopical projection images are modulated by a contrast transfer function (CTF) that prevents the calculation of three-dimensional reconstructions of biological complexes at high resolution from uncorrected images. We describe here an automated method for the accurate determination and correction of the CTF parameters defocus, twofold astigmatism and amplitude-contrast proportion from single-particle images. At the same time, the method allows the frequency-dependent signal decrease (B factor) and the non-convoluted background signal to be estimated. The method involves the classification of the power spectra of single-particle images into groups with similar CTF parameters; this is done by multivariate statistical analysis (MSA) and hierarchically ascending classification (HAC). Averaging over several power spectra generates class averages with enhanced signal-to-noise ratios. The correct CTF parameters can be deduced from these class averages by applying an iterative correlation procedure with theoretical CTF functions; they are then used to correct the raw images. Furthermore, the method enables the tilt axis of the sample holder to be determined and allows the elimination of individual poor-quality images that show high drift or charging effects.

Algorithms↗
PubMed2004

Targeting tumor necrosis factor-alpha in the therapy of psoriasis.

Tumor necrosis factor-alpha (TNF-alpha) plays a fundamental role in the initiation and persistence of skin inflammation in psoriasis. The best evidence of the essential activity of this cytokine in the pathogenesis of psoriasis came from the observation that selective TNF-alpha blockers are dramatically effective in the therapy of this disease. The TNF-alpha inhibitors, infliximab and etanercept, have been employed with success in moderate to severe psoriasis and in psoriatic arthritis in randomized controlled trials. Anti-TNF-alpha biologicals induce rapid disease resolution and long-lasting remission, suggesting that they may alter the natural course of the disease. Further studies are warranted to more precisely establish the biological bases of the action of anti-TNF-alpha agents, better define which subgroup of patients can benefit most from this treatment, and the modalities of combination therapy with other antipsoriatic agents. Many other TNF-alpha inhibitors have been developed but none of them has been yet used in the therapy of psoriasis. Major limitations to the use of selective TNF-alpha blockers include the reactivation of latent tuberculosis, the risk of opportunistic infections, the development of specific antibodies, which is associated with a reduced duration of response to treatment, and the high cost.

Antibodies, Monoclonal↗
PubMed2006

Identification of biologically active triterpenes and sterols present in hexane extracts from Miconia species using high-resolution gas chromatography.

In this work we describe the identification of the biologically active triterpenes and sterols present in the hexane extracts of six species of Miconia using gas chromatography. The main compounds present in these extracts are beta-amyrin, alpha-amyrin, and beta-sitosterol. The technique employed herein is shown to be a valuable and rapid tool for determining biologically active triterpenes and sterols present in non-polar extracts.

Chromatography, Gas↗
PubMed1986

Genetic studies of low-abundance human plasma proteins. I. Microheterogeneity of zinc-alpha 2-glycoprotein in biological fluids.

A high-resolution isoelectric focusing technique followed by immunoblotting has been utilized to determine the microheterogeneity of zinc-alpha 2-glycoprotein in a large number of plasma samples from U.S. Caucasians, Blacks, and Eskimos. With the exception of one Black individual, all samples were found to contain an invariant multiple-banded pattern which, after desialylation, was reduced to a single band, suggesting that the microheterogeneity observed is due to differences in the sialic acid content of a single protein product. The asialo forms of the variant sample consist of two distinct bands, consistent with the occurrence of a rare genetic variant at the zinc-alpha 2-glycoprotein structural locus. Unfortunately family studies were not feasible. In addition to plasma, the present technique has been applied to detection of zinc-alpha 2-glycoprotein microheterogeneity in amniotic fluid, saliva, and tears. The amniotic fluid pattern is identical to that present in plasma. However, the patterns observed in saliva and tears are different from each other as well as from that in plasma and could be controlled by separate loci.

Amniotic Fluid↗