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At least 217 records · Page 12Linked to original sources
PubMed2006

A quasi-Laue neutron crystallographic study of D-xylose isomerase.

The location of hydrogen atoms in enzyme structures can bring critical understanding of catalytic mechanism. However, whilst it is often difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (<1.0 A) resolution data available, neutron crystallography provides an experimental tool to directly localize hydrogen/deuterium atoms in biological macromolecules at resolution of 1.5-2.0 A. D-Xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) is a 43 kDa enzyme that catalyses the first reaction in the catabolism of D-xylose. Linearization and isomerization of D-xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data at 2.2 A resolution were collected at room temperature on a partially deuterated Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism. The neutron structure shows unambiguously that residue His 53 is doubly protonated at the active site of the enzyme. This suggests that the reaction proceeds through an acid catalyzed opening of the sugar ring, which is in accord with the mechanism suggested by Fenn et al. (Biochemistry 43(21): 6464-6474, 2004). This is the first report of direct observation of double protonation of His 53 and the first validation of the ring opening mechanism at the active site of D-xylose isomerase.

Aldose-Ketose Isomerases↗
PubMed2001

Determining the structure of biological macromolecules by transmission electron microscopy, single particle analysis and 3D reconstruction.

Single particle analysis and 3D reconstruction of molecules imaged by transmission electron microscopy have provided a wealth of medium to low resolution structures of biological molecules and macromolecular complexes, such as the ribosome, viruses, molecular chaperones and photosystem II. In this review, the principles of these techniques are introduced in a non-mathematical way, and single particle analysis is compared to other methods used for structural studies. In particular, the recent X-ray structures of the ribosome and of ribosomal subunits allow a critical comparison of single particle analysis and X-ray crystallography. This has emphasised the rapidity with which single particle analysis can produce medium resolution structures of complexes that are difficult to crystallise. Once crystals are available, X-ray crystallography can produce structures at a much higher resolution. The great similarities now seen between the structures obtained by the two techniques reinforce confidence in the use of single particle analysis and 3D reconstruction, and show that for electron cryo-microscopy structure distortion during sample preparation and imaging has not been a significant problem. The ability to analyse conformational flexibility and the ease with which time-resolved studies can be performed are significant advantages for single particle analysis. Future improvements in single particle analysis and electron microscopy should increase the attainable resolution. Combining single particle analysis of macromolecular complexes and electron tomography of subcellular structures with high-resolution X-ray structures may enable us to realise the ultimate dream of structural biology-a complete description of the macromolecular complexes of the cell in their different functional states.

Bacteria↗
PubMed1991

Platinum/iridium/carbon: a high-resolution shadowing material for TEM, STM and SEM of biological macromolecular structures.

Thin Pt/Ir/C coating films (1.5 nm) show a fine granularity and provide a high structural resolution in the transmission electron microscope (TEM) when applied to freeze-dried biological macromolecules. They keep their structure when exposed to atmospheric conditions, without the need of an additional stabilizing carbon layer, in contrast to conventional high-resolution shadowing materials such as Ta/W and Pt/C. However, the correct ratio of the components has turned out to be crucial. When evaporating Pt/Ir/C from the source electrode in an electron-beam-heated evaporator, the ratio of the three elements changes progressively, and, consequently, the properties of such films depend strongly on the mass that has been pre-evaporated. In this paper we present a quantitative analysis of the composition of Pt/Ir/C films by wavelength-dispersive X-ray analysis (WDX) undertaken in association with TEM experiments. We applied Pt/Ir/C shadowing to two regular biological test specimens, the phage T4 type III polyhead and the HPI-layer of Deinococcus radiodurans. It turns out that Pt/Ir/C films containing at least 25% C are three-dimensionally stable on the freeze-dried macromolecular samples. By the dramatically improved resolution power of the latest scanning electron microscopes (SEM) and the invention of the scanning tunnelling microscope (STM), two new surface-sensitive tools for the investigation of biological macromolecular structures became available. The Pt/Ir/C coating has proved to be well suited for STM and SEM imaging of freeze-dried biological structures because of its good electrical conductivity and its direct three-dimensional stability. We compare STM, SEM and TEM images of freeze-dried and Pt/Ir/C-coated polyheads.

Carbon↗
PubMed1977

Automated high-resolution gas chromatographic analysis of psychotropic drugs in biological fluids using open-tubular glass capillary columns. I. Determination of nomifensine in human plasma.

An automated high-resolution gas chromatographic method has been developed for the determination of low levels of the antidepressant psychotropic drug Nomifensine in human plasma. The drug is extracted from alkalinized plasma with diethyl ether and then back-extracted into an acidic aqueous phase. After subsequent extraction into diethyl ether the drug is analysed by gas-liquid chromatography as its heptafluorobutyrate derivative using an OV-101 open-tubular glass capillary column with a nitrogen-specific detector. The propyl and butyl analogues of Nomifensine are used as internal standards, added to the plasma before extraction. The method is accurate, specific and precise, and capable of measuring plasma concentrations down to a level of 2 ng/ml. A preliminary study of the pharmacokinetics of the drug, together with steady-state level measurements in normal individuals receiving therapeutic dosages of Nomifensine, has been made.

Adult↗
PubMed1975

High resolution electron microscope study of lattice images in biological apatites.

Lattice (Fourier) images of crystallites in human bone and teeth, and calcified atherosclerotic plaque were studied using high resolution transmission electron microscope techniques. The lattice images observed in the normal and diseased calcified tissue were compared with the images of synthetic hydroxyapatite crystallites.

Apatites↗
PubMed1998

Structure of a heparin-linked biologically active dimer of fibroblast growth factor.

The fibroblast growth factors (FGFs) form a large family of structurally related, multifunctional proteins that regulate various biological responses. They mediate cellular functions by binding to transmembrane FGF receptors, which are protein tyrosine kinases. FGF receptors are activated by oligomerization, and both this activation and FGF-stimulated biological responses require heparin-like molecules as well as FGF. Heparins are linear anionic polysaccharide chains; they are typically heterogeneously sulphated on alternating L-iduronic and D-glucosamino sugars, and are nearly ubiquitous in animal tissues as heparan sulphate proteoglycans on cell surfaces and in the extracellular matrix. Although several crystal structures have been described for FGF molecules in complexes with heparin-like sugars, the nature of a biologically active complex has been unknown until now. Here we describe the X-ray crystal structure, at 2.9 A resolution, of a biologically active dimer of human acidic FGF in a complex with a fully sulphated, homogeneous heparin decassacharide. The dimerization of heparin-linked acidic FGF observed here is an elegant mechanism for the modulation of signalling through combinatorial homodimerization and heterodimerization of the 12 known members of the FGF family.

Animals↗
PubMed1999

AFM tips: how sharp are they?

From both simple estimates and a 'blind' reconstruction based on cryo-AFM images of filamentous actin, we find that the radius of curvature at the apex of Si3N4 tips can be as small as 1 nm with a conical angle in the range 30 approximately 40 degrees, revealing a relatively high aspect ratio that is much greater than previously anticipated. Our results show that commercially available cantilevers are often sharp enough for routine high resolution imaging of biological materials, and suggest that factors other than an inherent blunt tip are probably responsible for frequent occurrences of poor resolution.

Actins↗
PubMed1997

Dual-axis tomography: an approach with alignment methods that preserve resolution.

Tomographic reconstructions of biological specimens are now routinely being generated in our high voltage electron microscope by tilting the specimen around two orthogonal axes. Separate tomograms are computed from each tilt series. The two tomograms are aligned to each other with general 3-D linear transformations that can correct for distortions between the two tomograms, thus preserving the inherent resolution of the reconstruction throughout its volume. The 3-D Fourier transforms of the two tomograms are then selectively combined to achieve a single tomogram. Unlike a single-axis tomogram, a dual-axis tomogram shows good resolution for extended features at any orientation in the plane of the specimen; it also has improved resolution in the depth of the specimen. Calculations indicate that the improvements available from double tilting and from tilting to higher angles are largely additive. Actual and model data were used to assess whether varying the increment between tilted views in proportion to the cosine of the tilt angle would allow a reduction in the number of pictures required to achieve a given resolution of reconstruction. Analysis by Fourier sector correlation indicated that the variable tilt increment improved the reconstruction in some respects but degraded it in others. A varying tilt increment thus does not give an unqualified improvement, at least when using back-projection algorithms for the reconstruction.

Animals↗
PubMed1996

Biological significance in forward and backward blocking: resolution of a discrepancy between animal conditioning and human causal judgment.

Similarities between Pavlovian conditioning in nonhumans and causal judgment by humans suggest that similar processes operate in these situations. Notably absent among the similarities is backward blocking (i.e., retrospective devaluation of a signal due to increased valuation of another signal that was present during training), which has been observed in causal judgment by humans but not in Pavlovian responding by animals. The authors used rats to determine if this difference arises from the target cue being biologically significant in the Pavlovian case but not in causal judgment. They used a sensory preconditioning procedure in Experiments 1 and 2, in which the target cue retained low biological significance during the treatment, and obtained backward blocking. The authors found in Experiment 3 that forward blocking also requires the target cue to be of low biological significance. Thus, low biological significance is a necessary condition for a stimulus to be vulnerable to blocking.

Animals↗
PubMed2004

High-resolution capillary electrophoresis of nitrite and nitrate in biological samples.

Nitrite and nitrate are widely used reporters of endogenous nitric oxide (NO) and nitric oxide synthase (NOS) activity, which are crucial for a broad spectrum of physiological and pathophysiological pathways. Because of the great variety in spatial expression and activity of NOS in animal tissues, a high-resolution analysis of nitrite/nitrate concentrations in very small biological samples, such as individual cells or homogeneous cell clusters, is required. A high-performance capillary zone electrophoresis (CZE) system, which includes a PrinCE-476 computerized capillary electrophoresis and Crystal-1000 conductivity detector, was optimized to analyze nitrite/nitrate concentrations in submicroliter samples of mammalian neuronal tissues and large individual cells of invertebrates. Solid-phase microextraction (SPME) and Isotachophoretic stacking (ITS) were used. The method is highly reproducible and yield excellent limits of detection (LODs): 8.9 nM (0.41 ppb) and 3.54 nM (0.22 ppb) for nitrite and nitrate, respectively, relative to undiluted samples.

Electrophoresis, Capillary↗
PubMed1991

Structural studies of membranes and surface layers up to 1,000 A thick using X-ray standing waves.

The X-ray standing wave (XSW) method, developed in the 1960s, was used originally to determine heavy atom positions in and on silicon and germanium single crystals. An X-ray standing wave generated by the interference of coherent incident and reflected beams excites X-ray fluorescence from the heavy atom, the intensity of which as a function of incident angle provides an indication of the atom's distance from the X-ray reflecting surface. The availability of X-ray mirrors and the ability to prepare layered synthetic microstructures has made possible the study of biologically relevant structures using the XSW technique on length scales of typically tens to hundreds of ångströms, allowing heavy atoms in such structures to be located with ångström or subångström resolution. Many model biological systems (such as Langmuir-Blodgett films, which mimic membranes) require access to still larger scales, but it is not obvious that an XSW will remain coherent over such length scales. Here we report studies of a lipid multilayer system using the XSW method, in which we have been able to locate the metal atoms in a zinc arachidate bilayer with ångström resolution at a distance of almost 1,000 A above the surface of a gold mirror. Our results indicate that the XSW technique should be useful for structural studies of supramolecular aggregates, receptor-ligand interactions and multi-membrane stacks, in which length scales of this order are encountered.

Arachidonic Acids↗
PubMed2000

Rapid and simultaneous high-performance liquid chromatography assay of polyamines and monoacetylpolyamines in biological specimens.

A rapid, resolutive and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for polyamines and acetylpolyamines by adopting pre-column derivatization with benzoyl chloride. In a single run lasting less than 15 min ten polyamines were separated as well as traces of benzoic acid, methylbenzoate and benzoic anhydride. These contaminants, produced during the derivatization reaction, were almost all eliminated by washing steps envisaged in the same procedure. This simple and sensitive method can be applied to routine determination of polyamines in biological samples. A fine application of this procedure to the determination of endogenous content of polyamines in chick embryo retina was reported.

Biogenic Polyamines↗
PubMed2006

Molecular recognition imaging and force spectroscopy of single biomolecules.

In recent years, considerable attention has focused on biological applications of the atomic force microscope (AFM), in particular on high-resolution imaging of individual biological molecules and on the measurement of molecular forces under near-physiological conditions. The detection of intermolecular forces in the piconewton range has paved the way to investigate details on structural parameters of the binding pockets and the energy landscapes of many biomolecular interactions. The capability of AFM to resolve nanometer-sized details, together with its force detection sensitivity, led to the development of molecular recognition imaging. By a combination of topographical imaging with force measurements, receptor sites are localized with nanometer accuracy. Topography and recognition of target molecules are thereby simultaneously mapped. Thus the AFM can identify specific components in a complex biological sample and retain its high resolution in imaging.

Aluminum Silicates↗
PubMed1999

Capillary electrophoresis of N-acetylneuraminic acid polymers and hyaluronic acid: correlation between migration order reversal and biological functions.

High-resolution analysis of polymers of N-acetylneuraminic acid and hyaluronic acid was performed by capillary electrophoresis in a buffer containing a neutral polymer. Both polysaccharides having more than 100 monosaccharide residues were well separated into their molecular species by capillary electrophoresis using a combination of a chemically modified capillary and a buffer containing poly(ethylene glycol) as an additive. During optimization of the separation conditions, small oligomers of both polysaccharides were observed to migrate in the reverse order of their molecular masses on the electropherograms. However, oligomers larger than pentamer and decamer for N-acetylneuraminic acid polymers and hyaluronic acid, respectively, migrated in the order of their molecular masses. We propose that these unusual migration patterns are closely related to the stereochemical structures and the oligomer migrating the fastest is the minimum unit that forms the regular three-dimensional structure required for the biological function.

Carbohydrate Sequence↗
PubMed1989

General principles of DNA repair in microorganisms and implications for future research.

The analysis of DNA repair and mutagenesis in organisms that have high resolution genetic and molecular biological systems has led, and will continue to lead, to major advances in our understanding of these processes. It is striking that almost all of the major insights highlighted in this chapter have been gained within the last 10 years and, in fact, several of the discoveries described here occurred during the actual writing of this report. It is worth reflecting that in the mid 1970s there was a widely held view that the essential elements of DNA repair in E. coli were well understood and, at that time, a considerable number of investigators shifted their focus to systems that did not offer the high resolution genetic and molecular biologic tools of E. coli and S. cerevisiae. The remarkable progress achieved over the past ten years underscores the limitations of that viewpoint. It is difficult, and probably unwise, to pinpoint specific issues in DNA repair in E. coli and S. cerevisiae as special targets for future research since so much remains to be learned. Most of the striking discoveries outlined in this chapter have come from skilled investigators using the full range of high resolution genetic, molecular biologic, and biochemical tools to address fundamental problems in DNA repair and mutagenesis. In most cases, it would have been impossible to have predicted the critical discoveries that have been made. For example, UV and chemical mutagenesis require the induction of a pair of genes, mediated by DNA-damage-stimulated proteolytic cleavage of a repressor and then the subsequent activation of one of these gene products by a mechanistically related proteolytic cleavage. In 1975 an hypothesis proposing such a mechanism for UV and chemical mutagenesis would have been dismissed as being utterly fanciful. Yet this is the current reality. As in the cases of the other findings detailed here, the key insights came not from the testing of fanciful hypotheses but rather from systematic scientific studies of fundamental problems. Funding in this area should then support inquiries of fundamental issues in DNA repair and mutagenesis by investigators who are skilled at using the full range of sophisticated experimental tools available for these organisms. It is worth noting that supporting this type of research will shed light not only on mechanisms of DNA repair and mutagenesis but also on more fundamental issues in the biology of living organisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Physiological↗
PubMed1998

Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors.

Liver fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSC), which proliferate during fibrotic liver injury. We have studied a model of spontaneous recovery from liver fibrosis to determine the biological mechanisms mediating resolution. Livers were harvested from rats at 0, 3, 7, and 28 d of spontaneous recovery from liver fibrosis induced by 4 wk of twice weekly intraperitoneal injections with CCl4. Hydroxyproline analysis and histology of liver sections indicated that the advanced septal fibrosis observed at time 0 (peak fibrosis) was remodeled over 28 d of recovery to levels close to control (untreated liver). alpha-Smooth muscle actin staining of liver sections demonstrated a 12-fold reduction in the number of activated HSC over the same time period with evidence of HSC apoptosis. Ribonuclease protection analysis of liver RNA extracted at each recovery time point demonstrated a rapid decrease in expression of the collagenase inhibitors TIMP-1 and TIMP-2, whereas collagenase mRNA expression remained at levels comparable to peak fibrosis. Collagenase activity in liver homogenates increased through recovery. We suggest that apoptosis of activated HSC may vitally contribute to resolution of fibrosis by acting as a mechanism for removing the cell population responsible for both producing fibrotic neomatrix and protecting this matrix from degradation via their production of TIMPs.

Animals↗
PubMed2006

Digital holographic microscopy with dual-wavelength phase unwrapping.

We apply the techniques of digital holography to obtain microscopic three-dimensional images of biological cells. The optical system is capable of microscopic holography with diffraction-limited resolution by projecting a magnified image of a microscopic hologram plane onto a CCD plane. Two-wavelength phase-imaging digital holography is applied to produce unwrapped phase images of biological cells. The method of three-wavelength phase imaging is proposed to extend the axial range and reduce the effect of phase noise. These results demonstrate the effectiveness of digital holography in high-resolution biological microscopy.

Algorithms↗
PubMed2000

Atomic force microscopy in structural biology: from the subcellular to the submolecular.

Atomic force microscopy (AFM) is capable of generating images within ranges of resolution that are of particular interest in biology. Although atomic resolution may not be possible with biological samples, a great deal of information can still be obtained from images that provide structures at a slightly lower level of resolution. The submolecular resolution images of bacteriorhodopsin and the chaperonin GroES, which revealed, respectively, individual loops and beta-turns, confirmed and complemented other structural investigations, while the molecular-level features in images of membrane-bound VacA, a cytotoxin from Helicobacter pylori, immediately suggested the possibility, subsequently proven, of channel-forming ability. A series of images with macromolecular resolution directly provided details on the mechanisms by which RNA polymerase nonspecifically translocates along DNA, and images with subcellular resolving power of erythrocytic cellular membranes showed, with unambiguous clarity, linear arrays of molecular complexes. In this review, we will describe some of the most biologically relevant findings that have been obtained with AFM within ranges of resolution from the submolecular to the molecular, and from the macromolecular to the subcellular. Furthermore, we will describe some of the sample conditions and imaging environments that are likely important to achieve a particular level of resolution.

Bacterial Proteins↗