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PubMed1980

White lines in L-edge x-ray absorption spectra and their implications for anomalous diffraction studies of biological materials.

We have measured high-resolution x-ray absorption spectra of lanthanide (Ln) and heavy transition metal complexes that display prominent narrow absorption peaks near the L2 and L3 absorption edges. The anomalous scattering factors (f' and f"), which are mathematically related to the absorption cross section, have correspondingly sharp changes in their magnitude within 5-10 eV of the absorption edge. Calculations of the magnitude of the change in f' and f" demonstrate that significant changes (on the order of 20 electrons in f') can be expected for these materials. These substantial changes in the anomalous scattering factors have applications to deriving structural information for macromolecules from x-ray diffraction studies. The magnitude of the changes indicate that the anomalous scattering technique is a powerful means of obtaining structural characteristics for macromolecules in single crystals, in solution, and in biological membranes.

Crystallography↗
PubMed1989

Non-apatitic environments in bone mineral: FT-IR detection, biological properties and changes in several disease states.

Resolution enhanced FT-IR spectroscopy shows that non-apatitic environments of phosphate and carbonate ions occur in bone mineral. The spectroscopic characteristics of these environments and their chemical and biological properties are reviewed. The potential effectiveness of FT-IR for the detailed study of bone mineral changes in bone disease is shown by analysis of several samples.

Animals↗
PubMed1999

Nuclear shape and nuclear matrix protein composition in prostate and seminal vesicles.

OBJECTIVES: The nucleus controls cell function and behavior. The nuclear matrix determines internal nuclear changes. Two-dimensional gel electrophoresis is the reference standard for the analysis of nuclear matrix protein (NMP) composition. Differences in NMP composition should therefore be reflected by changes in nuclear shape. We investigated the differences in NMP composition and nuclear morphometry of the prostate and seminal vesicles. Both tissues are androgen-dependent sex accessory organs with completely different biologic behavior. METHODS: High-resolution two-dimensional gel electrophoresis and silver staining were used to evaluate NMP composition from histologically normal prostate and seminal vesicle epithelial cells. Nuclear morphometry, performed using a computer-assisted image analysis system, described the distribution, variability, and extremes of nuclear shape. RESULTS: NMP composition analysis demonstrated that both tissues have a similar NMP composition, and tissue-specific NMPs that were consistently present in all specimens of each tissue could not be demonstrated. Nuclear morphometry showed a significantly greater heterogeneity in nuclear shape in the seminal vesicles than in the prostate. CONCLUSIONS: The striking similarity of the NMP composition demonstrates the close biologic relationship between prostate and seminal vesicle tissue. The similar NMP composition does not correlate with the marked alterations in nuclear shape and structure between these tissues. Therefore, nuclear morphometry may depict differences in the functional state of a similar set of NMPs, shown by two-dimensional gel electrophoresis, which may be responsible for the different biologic behavior of these tissues.

Antigens, Nuclear↗
PubMed2003

Genetics and conservation biology.

Conservation genetics encompasses genetic management of small populations, resolution of taxonomic uncertainties and management units, and the use of molecular genetic analyses in forensics and to understanding species' biology. The role of genetic factors in extinctions of wild populations has been controversial, but evidence now shows that they make important contributions to extinction risk. Inbreeding has been shown to cause extinctions of wild populations, computer projections indicate that inbreeding depression has important effects on extinction risk, and most threatened species show signs of genetic deterioration. Inappropriate management is likely to result if genetic factors are ignored in threatened species management.

Animals↗
PubMed1996

Crystal structure of a biologically inactive mutant of toxic shock syndrome toxin-1 at 2.5 A resolution.

Toxic shock syndrome toxin-1 (TSST-1) is one of a family of staphylococcal exotoxins recognized as microbial superantigens. The toxin plays a dominant role in the genesis of toxic shock in humans through a massive activation of the immune system. This potentially lethal illness occurs as a result of the interaction of TSST-1 with a significant proportion of the T-cell repertoire. TSST-1, like other superantigens, can bind directly to class II major histocompatibility (MHC class II) molecules prior to its interaction with entire families of V beta chains of the T-cell receptor (TCR). The three-dimensional structure of a mutant (His-135-Ala) TSST-1 was compared with the structure of the native (wild-type) TSST-1 at 2.5 A resolution. The replacement of His 135 of TSST-1 with an Ala residue results in the loss of T-cell mitogenicity and toxicity in experimental animals. This residue, postulated to be directly involved in the toxin-TCR interactions, is located on the major helix alpha 2, which forms the backbone of the molecule and is exposed to the solvent. In the molecular structure of the mutant toxin, the helix alpha 2 remains unaltered, but the His to Ala modification causes perturbations on the neighboring helix alpha 1 by disrupting helix-helix interactions. Thus, the effects on TCR binding of the His 135 residue could actually be mediated, wholly or in part, by the alpha 1 helix.

Bacterial Toxins↗
PubMed1981

A low-cost method for rapid transfer function measurements with direct application to biological impedance analysis.

The measurement of the linear transfer function of a biological system has found wide use in the characterization of the system's input/output properties, and in the separation and measurement of the properties of the different subelements that make up the system. Unfortunately sophisticated and expensive instrumentation has traditionally been required to make these measurements. In this paper, we present detailed design specifications of a low-cost instrument that is capable of yielding transfer function measurements with a high degree of accuracy and speed. The instrument is comprised of a pseudo-random binary sequence signal generator with precise date acquisition synchronization circuits, interfaced to a general-purpose mini-or microcomputer system common to many laboratory environments. The instrument is capable of measuring the transfer function of an arbitrary biological system up to a bandwidth of 8.3 KHz, with a frequency resolution of 425 points. In cases where the biological measurements are not contaminated with experimental noise, the transfer function can be determined in as little as 47 ms of data collection. In the case where experimental noise is present in the biological measurements, a simple averaging method is described which results in an effective increase in the signal-to-noise ratio, thereby yielding accurate transfer function estimates. The instrument is especially well suited to the measurement of transfer functions of biological systems, where experimental noise is a problem and where only limited time is available to acquire stable measurements.

Costs and Cost Analysis↗
PubMed1984

Analysis of leukotrienes, prostaglandins, and other oxygenated metabolites of arachidonic acid by high-performance liquid chromatography.

High-performance liquid chromatography procedures were developed which separate leukotrienes (LTs), hydroxy-fatty acids (HETEs), prostaglandins (PGs), the stable metabolite of prostacyclin (6-keto-PGF1 alpha), the stable metabolite of thromboxane A2 (TXB2), 12-hydroxyheptadecatrienoic acid (HHT), and arachidonic acid (AA). Two methods employing reverse-phase columns are described. One method uses a radial compression system, the other a conventional steel column. Both systems employ methanol and buffered water as solvents. The radial compression system requires 60 min for separation of the AA metabolites, while the conventional system requires 100 min. Both methods provide good separation and recovery of 6-keto-PGF1 alpha, TXB2, PGE2, PGF2 alpha, PGD2, LTC4, LTB4, LTD4, LTE4, HHT, 15-, 12-, and 5-HETE; and AA. The 5S,12S-dihydroxy-6-trans, 8-cis, 10-trans, 14-cis-eicosatetraenoic acid (5S,12S-diHETE), a stereoisomer of LTB4, coelutes with LTB4. To determine the applicability of the methods to biologic systems, AA metabolism was studied in two models, guinea pig lung microsomes and rat alveolar macrophages. Both HPLC systems demonstrated good recovery and resolution of eicosanoids from the two biological systems. A simple evaporation technique for HPLC sample preparation, which avoids the use of chromatographic and other time-consuming methodology, is also described.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed2004

Equol, a natural estrogenic metabolite from soy isoflavones: convenient preparation and resolution of R- and S-equols and their differing binding and biological activity through estrogen receptors alpha and beta.

Equol is a metabolite produced in vivo from the soy phytoestrogen daidzein by the action of gut microflora. It is known to be estrogenic, so human exposure to equol could have significant biological effects. Equol is a chiral molecule that can exist as the enantiomers R-equol and S-equol. To study the biological activity of racemic (+/-)-equol, as well as that of its pure enantiomers, we developed an efficient and convenient method to prepare (+/-)-equol from available isoflavanoid precursors. Furthermore, we optimized a method to separate the enantiomers of equol by chiral HPLC, and we studied for the first time, the activities of the enantiomers on the two estrogen receptors, ERalpha and ERbeta. In binding assays, S-equol has a high binding affinity, preferential for ERbeta (K(i)[ERbeta]=16 nM; beta/alpha=13 fold), that is comparable to that of genistein (K(i)[ERbeta]=6.7 nM; beta/alpha=16), whereas R-equol binds more weakly and with a preference for ERalpha (K(i)[ERalpha]=50 nM; beta/alpha=0.29). All equol isomers have higher affinity for both ERs than does the biosynthetic precursor daidzein. The availability and the in vitro characterization of the equol enantiomers should enable their biological effects to be studied in detail.

Antineoplastic Agents↗
PubMedDate not supplied

[Lighting of biological objects during their visual recording by super thin rigid endoscopes].

The paper deals with the effects of illumination intensity levels on perception conformality and colour saturation in the image of a biological object. Reveals the impact of the optical parameters of an endoscope, the parameters characterizing the biological object and its illumination intensity on the resolution of the optic system of observer's eyes--an endoscope. The biological object illumination intensity range recommended in its visual recording by using rigid endoscopes is defined.

Color↗
PubMed2001

NASA/American Cancer Society High-Resolution Flow Cytometry Project-I.

BACKGROUND: The NASA/American Cancer Society (ACS) flow cytometer can simultaneously analyze the electronic nuclear volume (ENV) and DNA content of cells. This study describes the schematics, resolution, reproducibility, and sensitivity of biological standards analyzed on this unit. METHODS: Calibrated beads and biological standards (lymphocytes, trout erythrocytes [TRBC], calf thymocytes, and tumor cells) were analyzed for ENV versus DNA content. Parallel data (forward scatter versus DNA) from a conventional flow cytometer were obtained. RESULTS: ENV linearity studies yielded an R value of 0.999. TRBC had a coefficient of variation (CV) of 1.18 +/- 0.13. DNA indexes as low as 1.02 were detectable. DNA content of lymphocytes from 42 females was 1.9% greater than that for 60 males, with a noninstrumental variability in total DNA content of 0.5%. The ENV/DNA ratio was constant in 15 normal human tissue samples, but differed in the four animal species tested. The ENV/DNA ratio for a hypodiploid breast carcinoma was 2.3 times greater than that for normal breast tissue. CONCLUSIONS: The high-resolution ENV versus DNA analyses are highly reliable, sensitive, and can be used for the detection of near-diploid tumor cells that are difficult to identify with conventional cytometers. ENV/DNA ratio may be a useful parameter for detection of aneuploid populations.

American Cancer Society↗
PubMed2002

High resolution flow cytometric analysis of electronic nuclear volume and DNA content in normal and abnormal human tissue.

BACKGROUND: The NPE Analyzer flow cytometer can simultaneously analyze the electronic nuclear volume (ENV) and DNA content of cells. This study describes the schematics, resolution, reproducibility, and sensitivity of biological standards analyzed on this unit. METHODS: Calibrated beads and biological standards (lymphocytes, trout erythrocytes [TRBC], calf thymocytes, and tumor cells) were analyzed for ENV versus DNA content. Parallel data (forward scatter versus DNA) from a conventional flow cytometer were obtained. RESULTS: ENV linearity studies yielded an R value of 0.999. TRBC had a coefficient of variation (CV) of 1.18 +/- 0.13. DNA indexes as low as 1.02 were detectable. DNA content of lymphocytes from 42 females was 1.9% greater than that for 60 males, with a noninstrumental variability in total DNA content of 0.5%. The ENV/DNA ratio was constant in 15 normal human tissue samples, but differed in the four animal species tested. The ENV/DNA ratio for a hypodiploid breast carcinoma was 2.3 times greater than that for normal breast tissue. CONCLUSIONS: The high-resolution ENV versus DNA analyses are highly reliable, sensitive, and can be used for the detection of near-diploid tumor cells that are difficult to identify with conventional cytometers. ENV/DNA ratio may be a useful parameter for detection of aneuploid populations.

Biomarkers↗
PubMed2000

Characterization of a biological detector cell for quantitation of inositol 1,4,5-trisphosphate.

Prior strategies to measure inositol 1,4,5-trisphosphate (IP(3)) in single cells either have been qualitative or have had a limited spatial resolution. Capillary electrophoresis combined with a biological detector cell has been used to quantitate IP(3) in small regions of a Xenopus oocyte. To improve the detection limits of this method, we elucidated the experimental parameters which influenced the sensitivity and reliability of the IP(3)-detector cell coupled to capillary electrophoresis. The variables which influenced the detector cell were the magnitude of the voltage drop across the detector cell, the duration of this voltage drop, the direction of fluid flow in the capillary, the concentration of free Ca(2+) around the detector cell, and the presence of protease inhibitors during permeabilization of the detector cell. For the sample volumes imposed by the capillary diameter, the detector cell acted primarily as an IP(3) mass detector rather than a concentration detector. Characterization of the experimental variables influencing the sensitivity and reliability of this detector cell has the potential to enhance other analyte measurements performed by mating capillary electrophoresis with a biological detector cell.

Animals↗
PubMed2003

Nanosecond time-resolved crystallography of photo-induced species: case study and instrument development for high-resolution excited-state single-crystal structure determination.

This work describes one of the first stages in the development of time-resolved photo-induced small-molecule single-crystal diffraction, whereby transient electron density perturbations, with lifetimes down to the nanosecond level, can be resolved at the atomic level. Knowledge of such ephemeral electronic effects is likely to yield key information regarding the origins of certain important physical properties, e.g. luminescent and non-linear optical effects, since it will allow the dynamics of electron density to be identified and quantified, and it is this that underpins such phenomena in a given molecule. The experimental methodology employs phase-locking pump-probe techniques such that the inherent time-structure of a synchrotron X-ray beam (nanoseconds) is harnessed and time-gated in-phase with a femtosecond laser. The resultant beams, made coincident on the crystal in a periodic manner, and a diffraction pattern are recorded as a function of the Bragg angle, theta. Such technology is based upon the pioneering work carried out in sub-nanosecond time-resolved crystallography of macromolecular biological moieties (non-atomic resolution) at the ESRF, although one crucial difference here is the use of monochromatic irradiation and oscillatory motion rather than Laue 'snapshot' methodology, so that atomic resolution is possible. The experimental details of a case study conducted on ID9 at the ESRF, France, are described, whereby the feasibility of the excited-state structure determination of a luminescent rhenium carbene complex, [HNCH2CH2NHCRe(2,2'-bipyridine)(CO)3]Br, is realised. Key experimental parameters that are required for the success of such an experiment are discussed in the light of this study, together with other feasibility work conducted at the SRS, UK, and in the laboratory. Plans, designs and tests for the implementation of this technique in the UK, first at the SRS, and then at DIAMOND, the forthcoming UK synchrotron, are described, in particular with reference to the world-leading potential that DIAMOND could lend toward the development of this technique.

Journal Article↗
PubMed2005

Structure-function relationships in plant phenylpropanoid biosynthesis.

Plants, as sessile organisms, evolve and exploit metabolic systems to create a rich repertoire of complex natural products that hold adaptive significance for their survival in challenging ecological niches on earth. As an experimental tool set, structural biology provides a high-resolution means to uncover detailed information about the structure-function relationships of metabolic enzymes at the atomic level. Together with genomic and biochemical approaches and an appreciation of molecular evolution, structural enzymology holds great promise for addressing a number of questions relating to secondary or, more appropriately, specialized metabolism. Why is secondary metabolism so adaptable? How are reactivity, regio-chemistry and stereo-chemistry steered during the multi-step conversion of substrates into products? What are the vestigial structural and mechanistic traits that remain in biosynthetic enzymes during the diversification of substrate and product selectivity? What does the catalytic landscape look like as an enzyme family traverses all possible lineages en route to the acquisition of new substrate and/or product specificities? And how can one rationally engineer biosynthesis using the unique perspectives of evolution and structural biology to create novel chemicals for human use?

Biological Evolution↗
PubMed1990

Kingdom Animalia: the zoological malaise from a microbial perspective.

Pain and cognitive dissonance abounds amongst biologists: the plant-animal, botany-zoology wound has nearly healed and the new gash--revealed by department and budget reorganizations--is "molecular" vs. "organismic" biology. Here I contend that resolution of these tensions within zoology requires that an autopoietic-gaian view replace a mechanical-neodarwinian perspective; in the interest of brevity and since many points have been discussed elsewhere, rather than develop detailed arguments I must make staccato statements and refer to a burgeoning literature. The first central concept is that animals, all organisms developing from blastular embryos, evolved from single protist cells that were unable to reproduce their undulipodia. The second points to the usefulness of recognizing the analogy between cyclically established symbioses and meiotic sexuality.

Animal Population Groups↗
PubMed2000

Assembly and activation of site-specific recombination complexes.

Site-specific recombination is responsible for a broad range of biological phenomena, including DNA inversion, resolution of transposition intermediates, and the integration and excision of bacteriophage genomes. Integration of mycobacteriophage L5 is catalyzed by a phage-encoded integrase with recombination occurring between specific attachment sites on the phage and mycobacterial chromosomes (attP and attB, respectively). Although some site-specific recombination systems simply involve binding of the recombinase to the sites of strand exchange, synapsis, and recombination, phage systems typically require the assembly of higher-order structures within which the recombinational potential of integrase is activated. The requirement for these structures derives from the necessity to regulate the directionality of recombination-either integration or excision-which must be closely coordinated with other aspects of the phage growth cycles. We show herein that there are multiple pathways available for the assembly of L5 recombination complexes, including the early synapsis of the attP and attB DNAs. This process is in contrast to the model for lambda integration and illustrates the different usage of molecular machineries to accomplish the same biological outcome.

DNA, Bacterial↗