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PubMed2002

The catalytic pathway of horseradish peroxidase at high resolution.

A molecular description of oxygen and peroxide activation in biological systems is difficult, because electrons liberated during X-ray data collection reduce the active centres of redox enzymes catalysing these reactions. Here we describe an effective strategy to obtain crystal structures for high-valency redox intermediates and present a three-dimensional movie of the X-ray-driven catalytic reduction of a bound dioxygen species in horseradish peroxidase (HRP). We also describe separate experiments in which high-resolution structures could be obtained for all five oxidation states of HRP, showing such structures with preserved redox states for the first time.

Catalysis↗
PubMed2000

Dietary essential fatty acids, long-chain polyunsaturated fatty acids, and visual resolution acuity in healthy fullterm infants: a systematic review.

BACKGROUND: Biologically active neural tissue is rich in docosahexaenoic acid (DHA), an omega-3 long-chain polyunsaturated fatty acid (LCPUFA). We conducted a systematic review to examine the nature of discordant results from studies designed to test the hypothesis that dietary DHA leads to better performance on visually-based tasks in healthy, fullterm infants. We also conducted a meta-analysis to derive combined estimates of behavioral- and electrophysiologic-based visual resolution acuity differences and sample sizes that would be useful in planning future research. STUDY DESIGN AND METHODS: Twelve empirical studies on LCPUFA intake during infancy and visual resolution acuity were identified through bibliographic searches, examination of monograph and review article reference lists, and written requests to researchers in the field. Works were reviewed for quality and completeness of information. Study design and conduct information was extracted with a standardized protocol. Acuity differences between groups consuming a source of DHA and groups consuming DHA-free diets were calculated as a common outcome from individual studies; this difference score was evaluated against a null value of zero and then used, with the method of DerSimonian and Laird (Meta-analysis in clinical trials. Control Clin Trials 1986;7:177-188), to derive combined estimates of visual resolution acuity differences within seven age categories. RESULTS OF RANDOMIZED COMPARISONS: The combined visual resolution acuity difference measured with behaviorally based methods between DHA-supplemented formula fed groups and DHA-free formula fed groups is 0.32+/-0.09 octaves (combined difference+/-S.E.M., P=0.0003) at 2 months of age. The direction of this value indicates higher acuity in DHA-fed groups. RESULTS OF NON-RANDOMIZED STUDY DESIGNS: The combined visual resolution acuity difference measured with behaviorally based methods between human milk fed groups and DHA-free formula fed groups is 0.49+/-0.09 octaves (P< or =0.000001) at 2 months of age and 0.18+/-0.08 octaves (P=0.04) at 4 months of age. Acuity differences for electrophysiologic-based measures are also greater than zero at 4 months (0.37+/-0.16 octaves, P=0.02). CONCLUSION: Some aspect of dietary n-3 intake is associated with performance on visual resolution acuity tasks at 2, and possibly, 4 months of age in healthy fullterm infants. Whether n-3 intake confers lasting advantage in the development of visually based processes is still in question.

Bottle Feeding↗
PubMed2004

Isotopic variations of Zn in biological materials.

Variations in the isotopic composition of Zn present in various biological materials were determined using high-resolution multicollector inductively coupled plasma mass spectrometry (MC-ICPMS), following digestion and purification by anion exchange chromatography. To correct for differences in instrumental mass discrimination effects between samples and standards, Cu was employed as an elemental spike. Complementary analyses of Zn separates by sector field ICPMS instruments revealed that the concentrations of the majority of potentially interfering elements were reduced to negligible levels. Residual spectral interferences resulting from (35)Cl(16)O(2)(+), (40)Ar(14)N(2)(+), and (40)Ar(14)N(16)O(+) could be instrumentally resolved from the (67)Zn, (68)Zn, and (70)Zn ion beams, respectively, during measurement by MC-ICPMS. The only other observed interference in the Cu and Zn mass range that could not be effectively eliminated by high-resolution multicollection resulted from (35)Cl(2)(+), necessitating modification of the sample preparation procedure to allow accurate (70)Zn detection. Complete duplication of the entire analytical procedure for human whole blood and hair, as well as bovine liver and muscle, provided an external reproducibility of 0.05-0.12 per thousand (2sigma) for measured delta(66/64)Zn, delta(67/64)Zn, and delta(68/64)Zn values, demonstrating the utility of the method for the precise isotopic analysis of Zn in biological materials. Relative to the selected Zn isotopic standard, delta(66/64)Zn values for biological samples varied from -0.60 per thousand in human hair to +0.56 per thousand in human whole blood, identifying the former material as the isotopically lightest Zn source found in nature to date.

Animals↗
PubMed1988

High resolution 31P NMR of extracted phospholipids.

The common phospholipids from biological sources were quantitated using phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy in conjunction with an analytical reagent composed of two parts: 1) 2 ml of reagent chloroform in which was dissolved 0.01-100 mg of crude tissue lipid extracted from tissue sources using chloroform-methanol 2:1, the extract having been washed with 0.2 vol. of 0.1 M KCl; 2) 1 ml of an aqueous methanol reagent composed of one part 0.2 M (ethylenedinitrilo)-tetraacetic acid in D2O titrated to pH 6 with CsOH and four parts of reagent methanol. In a magnetic field of 11.75 Tesla, the extracted phospholipids yield narrow signals (1.8-3.2 Hz at half-height), corresponding to each generic species, e.g., phosphatidylcholines, phosphatidylethanolamines, etc., permitting resolution among the various phospholipid families and their lyso and plasmalogen derivatives. The reagent permits assays of high precision and accuracy using a modest amount of NMR spectrometer time (ca. 15 min/assay). The procedures described, which are compared to high-performance liquid chromatography, are convenient for the routine analysis of phospholipids from biological sources.

Animals↗
PubMed1997

Crystal structure of pentalenene synthase: mechanistic insights on terpenoid cyclization reactions in biology.

The crystal structure of pentalenene synthase at 2.6 angstrom resolution reveals critical active site features responsible for the cyclization of farnesyl diphosphate into the tricyclic hydrocarbon pentalenene. Metal-triggered substrate ionization initiates catalysis, and the alpha-barrel active site serves as a template to channel and stabilize the conformations of reactive carbocation intermediates through a complex cyclization cascade. The core active site structure of the enzyme may be preserved among the greater family of terpenoid synthases, possibly implying divergence from a common ancestral synthase to satisfy biological requirements for increasingly diverse natural products.

Alkyl and Aryl Transferases↗
PubMed1991

[Medulloblastoma: towards new prognostic factors].

Current therapeutic protocols for medulloblastoma combining, with surgery, radiation therapy and chemotherapy allow 5-year survival rates of over 50%. However these therapies induce mainly in children long-term adverse effects, which produce a therapeutic dilemma emphasizing the need for reliable prognostic factors in order to adapt the treatment modalities to the degree of the tumor's aggressiveness. Contradictory results have been reported concerning the conventional clinical and histological prognostic factors in medulloblastoma. Recent development and simplification of cell biology technologies could now help in the resolution of this issue. The aim of this review is to present some new prognostic factors available from these advances, and to discuss their potential usefulness in the field of medulloblastoma: The measurement of the proliferative potential of tumors using thymidine analogues such as bromodeoxyuridine (BUdR), or the monoclonal antibody Ki-67 is promising. The flow cytometric determination of D.N.A. content in medulloblastomas appears to show a correlation of diploidy with a worse prognosis than aneuploidy. Modern cytogenetic and molecular biology techniques are permitting the current assessment of amplification and overexpression of oncogenes, or the presence of deletions, as prognostic factors in medulloblastoma. The study of the cellular phenotype and particularly the search for differentiation markers has not yet led to clear-cut results from a prognostic viewpoint, but further advances are to be expected in this field, thanks to the development of more specific monoclonal antibodies. The investigation of the tumoral stroma and metabolism is very promising too. These novel approaches to prognostic factors in medulloblastoma should allow a better classification and management of these tumors in the near future.

Antibodies, Monoclonal↗
PubMed1991

Applications of confocal scanning optical microscopy to dentistry.

Confocal optical microscopy is now a well recognised technique in the fields of biological and materials science. This type of light microscope can be considered as being midway between optical and electron microscopy. Confocal or scanning optical microscopes can make high resolution, thin, optical sections within semitransparent samples such as biological tissues. Surface images of samples can be produced which are similar in character to those of the SEM, but without many of the problems of specimen preparation. The improved resolution and removal of out-of-focus blur allows much more information to be gained from fluorescence microscopy techniques, with the images capable of 3-D reconstruction of the sample. There are basically two types of confocal optical microscope: the laser scanning type (CLSM) and the real-time direct view of tandem scanning microscopes (TSM). The former are best suited to immunofluorescence microscopy, whilst the latter are more appropriate for high-speed reflection imaging, having originally been developed for in vivo microscopy.

Dentistry, Operative↗
PubMed1998

Cell biological applications of scanning near-field optical microscopy (SNOM).

Scanning near-field optical microscopy (SNOM) yields high-resolution topographic and optical images and is an important technique for visualizing biological systems. We summarize the literature on SNOM of biological systems and present some of our recent applications in cellular biology. These include studies of: i) the binding of fluorescently conjugated lectins to cell surface glycoproteins on 3T3 Balb/c cells, ii) molecular interactions by fluorescence resonance energy transfer using photobleaching techniques, and iii) green fluorescent protein (GFP) expressed in bacteria.

Animals↗
PubMed2006

High resolution 1H detected 1H,13C correlation spectra in MAS solid-state NMR using deuterated proteins with selective 1H,2H isotopic labeling of methyl groups.

MAS solid-state NMR experiments applied to biological solids are still hampered by low sensitivity and resolution. In this work, we employ a deuteration scheme in which individual methyl groups are selectively protonated. This labeling scheme allows the acquisition of proton carbon correlation spectra with a resolution comparable to that in solution-state NMR experiments. We observe an increase in resolution by a factor of 10-15 compared to standard heteronuclear correlation experiments using PMLG for 1H,1H dipolar decoupling in the indirect dimension. At the same time, the full sensitivity of the proton-based experiment is retained. In comparison to the heteronuclear detected version of the experiment, a gain in sensitivity of a factor of approximately 4.7 is achieved.

Animals↗
PubMed2002

Microspectroscopy and imaging in the THz range using coherent CW radiation.

A novel THz near-field spectrometer is presented which allows the performance of biological and medical studies with high spectral resolution combined with a spatial resolution down to lambda/100. In the setup an aperture much smaller than the used wavelength is placed in the beam very close to the sample. The sample is probed by the evanescent wave behind the aperture. The distance is measured extremely accurately by a confocal microscope. We use monochromatic sources which provide powerful coherent cw radiation tuneable from 50 GHz up to 1.5 THz. Transmission and reflection experiments can be performed which enable us to study solids and molecules in aqueous solution. Examples for spectroscopic investigations on biological tissues are presented.

Adipose Tissue↗
PubMed2005

Electrical conductivity images of biological tissue phantoms in MREIT.

We present cross-sectional conductivity images of two biological tissue phantoms. Each of the cylindrical phantoms with both diameter and height of 140 mm contained chunks of biological tissues such as bovine tongue and liver, porcine muscle and chicken breast within a conductive agar gelatin as the background medium. We attached four recessed electrodes on the sides of the phantom with equal spacing among them. Injecting current pulses of 480 or 120 mA ms into the phantom along two different directions, we measured the z-component Bz of the induced magnetic flux density B=(Bx, By, Bz) with a magnetic resonance electrical impedance tomography (MREIT) system based on a 3.0 T MRI scanner. Using the harmonic Bz algorithm, we reconstructed cross-sectional conductivity images from the measured Bz data. Reconstructed images clearly distinguish different tissues in terms of both their shapes and conductivity values. In this paper, we experimentally demonstrate the feasibility of the MREIT technique in producing conductivity images of different biological soft tissues with a high spatial resolution and accuracy when we use a sufficient amount of the injection current.

Algorithms↗
PubMed2003

A new neutron single-crystal diffractometer dedicated for biological macromolecules (BIX-4).

A new neutron single-crystal diffractometer (BIX-4) has been constructed at 1G-B port of JRR-3M in JAERI. Since at 1G-B port another diffractometer for biology, BIX-3, and a high-resolution powder diffractometer (HRPD) coexist, the monochromator house needed to be reconstructed. The main architecture of BIX-4 is based on that of BIX-3. BIX-4 uses an elastically-bent perfect-Si crystal monochromator and neutron imaging plates as BIX-3. In addition, several optimizations of the monochromator and modifications from previous BIX-3 are carried out. The final gain of the neutron intensity at the detector position is estimated to be 2.5 times larger than previous BIX-3. That higher performance increases the opportunities to apply neutron crystallography to biological macromolecules which give only weak reflections and/or small crystals.

Biopolymers↗
PubMed1984

Imaging mammalian tissues and organs using laser collimated transillumination.

Images of mammalian organs were built up using a Laser collimated transillumination device equipped with a bidirectional scanning setup. A microcomputer was used to run the scanning process, acquisition of detected signals and images restitution. The images featured a satisfactory resolution of optical discontinuities on both sample surfaces. Light scattering within biological tissues restricted the spatial discrimination encountered with geometrical selection approach. By combining space and time resolution at various wavelengths, the production of an efficient tomospectroscopy could be envisaged.

Animals↗
PubMed1981

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.

Acetylcholine↗
PubMed2001

High-resolution analysis of tomato leaf elongation: the application of novel time-series analysis techniques.

This paper demonstrates the use of a novel suite of data-based, recursive modelling techniques for the investigation of biological and other time-series data, including high resolution leaf elongation. The Data-Based Mechanistic (DBM) modelling methodology rejects the common practice of empirical curve fitting for a more objective approach where the model structure is not assumed a priori, but instead is identified directly from the data series in a stochastic form. Further, this novel approach takes advantage of the latest techniques in optimal recursive estimation of non-stationary and non-linear time-series. Here, the utility and ease of use of these techniques is demonstrated in the examination of two time-series of leaf elongation in an expanding leaf of tomato (Lycopersicon esculentum L. cv. Ailsa Craig) growing in a root pressure vessel (RPV). Using this analysis, the component signals of the elongation series are extracted and considered in relation to physiological processes. It is hoped that this paper will encourage the wider use of these new techniques, as well as the associated Data-Based Mechanistic (DBM) modelling strategy, in analytical plant physiology.

Algorithms↗
PubMed2000

The need for chromatographic and mass resolution in liquid chromatography/tandem mass spectrometric methods used for quantitation of lactones and corresponding hydroxy acids in biological samples.

Because of the potential in-source conversion between a lactone and the corresponding hydroxy acid, it has been recognized that a liquid chromatography/tandem mass spectrometric (LC/MS/MS) method developed for quantitation of a lactone drug in the presence of its hydroxy acid metabolite (or vice versa) must incorporate chromatographic separation between the two compounds, unless in-source conversion between the two compounds has been eliminated by the appropriate selection of the LC/MS/MS parameters. We now report that chromatographic separation between a lactone and its hydroxy acid will be required under certain LC/MS/MS conditions used even in the absence of in-source conversion. This is due to the fact that the 18-mass-unit difference between a lactone and its hydroxy acid is, by coincidence, different by only one mass unit from the 17-mass-unit difference between the [M + H](+) and [M + NH(4)](+) ions of the lactone or the hydroxy acid. Thus, the [M + H](+) ion of a hydroxy acid is higher than the [M + NH(4)](+) ion of its lactone by only one mass unit. Therefore, in a method developed for quantitation of a hydroxy acid drug utilizing a selected-ion-monitoring (SRM) scheme that incorporates its [M + H](+) ion as the precursor ion, the quantitation would be inaccurate due to the interference by the contribution of the A + 1 isotope response from the [M + NH(4)](+) ion of the lactone metabolite present in the sample, unless there is a chromatographic separation between the two compounds. This is true even if Q1 is operated under a unit-mass resolution. The implication of this type of interference, arising from the presence of both the [M + H](+) and [M + NH(4)](+) ions of a drug and its metabolite, to the selection of LC and MS conditions (including mass resolution) will be discussed using the data obtained with a model lactone drug and its hydroxy acid metabolite.

Chromatography, High Pressure Liquid↗
PubMed1996

Absence of splenic latency in murine gammaherpesvirus 68-infected B cell-deficient mice.

Murine gammaherpesvirus 68 (MHV-68) is a natural pathogen of mice which causes an acute lung infection and establishes a latent infection in B lymphocytes. In this paper we describe the infection in transgenic B cell-deficient (muMT) mice, to determine whether a latent infection can be established in a mouse lacking circulating B lymphocytes. Little difference was observed in the acute lung infection, although there was a slight delay in virus clearance in the muMT mice. This indicates that antiviral antibody is of little importance in the resolution of the lung infection. Neither free nor latent virus could be detected in the spleen in the muMT mice. In addition, these mice did not develop MHV-68-induced splenomegaly. These data suggest that within the lymphoid compartment B lymphocytes are the sole reservoir for MHV-68 infection in vivo, confirming earlier work which identified B cells as the site of latent infection based on cell fractionation studies. In addition, our study shows that CD4-driven lymphocyte expansion leading to splenomegaly is dependent on the presence of MHV-68-infected B cells in the spleen. Although no free virus was detected (using conventional biological assays) in the lung after the resolution of the acute infection, MHV-68 genome was detected in the lungs of both control and muMT mice by PCR analysis. This suggests that cells in the lung may act as a reservoir of latent virus which is independent of the B lymphocyte infection.

Animals↗