The application of mass spectrometry in the structural elucidation of glycosphingolipids

Mass spectrometry has been successfully applied to the analysis of permethylated glycosphingolipids, with and without reduction, as well as of permethylated gangliosides after reduction and silylation. The results obtained by several groups of workers are reviewed. From the data available it can be stated that, with the aid of mass spectrometry, conclusive evidence may be obtained concerning the carbohydrate sequence as far as the typo of sugar is concerned such as hexose, deoxyhexose, hexosamine, and neuraminic acid residues. Branching points can be recognized, and specific fragmentation products allow the differentiation between 1,3 and 1,4-substituted hexosamines of blood-group-active glycosphingolipids (type 1 and type 2 chains). The ceramide residue is documented by several characteristic ions that allow determination not only of sphingosine bases and fatty acid components but also of individual ceramide molecular species. Valuable structural information can thus be obtained to a certain extent also for mixtures of glycosphingolipids composed of different carbohydrate chains or different ceramide residues.     

Biologically Inspired Information Processing and Synchronization in Ensembles of Non-Identical Threshold-Potential Nanostructures

Nanotechnology produces basic structures that show a significant variability in their individual physical properties. This experimental fact may constitute a serious limitation for most applications requiring nominally identical building blocks. On the other hand, biological diversity is found in most natural systems. We show that reliable information processing can be achieved with heterogeneous groups of non-identical nanostructures by using some conceptual schemes characteristic of biological networks (diversity, frequency-based signal processing, rate and rank order coding, and synchronization). To this end, we simulate the integrated response of an ensemble of single-electron transistors (SET) whose individual threshold potentials show a high variability. A particular experimental realization of a SET is a metal nanoparticle-based transistor that mimics biological spiking synapses and can be modeled as an integrate-and-fire oscillator. The different shape and size distributions of nanoparticles inherent to the nanoscale fabrication procedures result in a significant variability in the threshold potentials of the SET. The statistical distributions of the nanoparticle physical parameters are characterized by experimental average and distribution width values. We consider simple but general information processing schemes to draw conclusions that should be of relevance for other threshold-based nanostructures. Monte Carlo simulations show that ensembles of non-identical SET may show some advantages over ensembles of identical nanostructures concerning the processing of weak signals. The results obtained are also relevant for understanding the role of diversity in biophysical networks.     

Molecular approach to ayurveda

In ayurvedic system of medicine, it is considered that a living system is made of panch-mahabuta, in the form of Vata, pitta and kapha at the physical level and satwa, raja and tama at the mental level. This covers the psychosomatic constitution and commonly known as the Tridosh theory. The imbalance in these body humours is the basic cause of any type of disease manifestation. Till date, several objective parameters have been proposed to monitor the level of these basic humours but none of them is complete. In this exercise, now it is proposed to consider free radical theory of diseases as one of the objective parameters. To be more specific, vata can be monitored in terms of membrane bound signal transduction, pitta as the process of phosphorylation and de-phosphorylation of different proteins (signalling moieties and enzymes) and kapha can be viewed as the degree of gene expression as protein synthesis. This can be correlated with the ojas of the body or total body defence mechanism.     

Current methodological refinements for the acquisition of life history information in fishes: paradigms from pan-oceanic billfishes.

1. Many factors contribute to hinder progress in estimating and verifying age, growth, and life history strategies of billfishes. It is necessary to discern growth of an individual fish to circumvent problems in substantiating age, growth, longevity and age at maturity. These biological characteristics allow us to discover what life history periods are critical to survival and reproduction. 2. Two theoretical approaches may be considered for studying the age of billfish: Age may be delineated in terms of the passage of time (chronological age), or may be examined as the progression of physiological development (physiological age). 3. Hard tissues such as otoliths, spines, vertebrae and post-mitotic tissues contain a plethora of biological and ecological information documenting a fish's chronological and physiological age and individual life history. In calcified tissues, this information can be obtained when the proper techniques are used to investigate the structural and chemical components of billfish hard parts. Chemical analyses of stable isotopes and elemental concentrations in calcified tissues suggest that calcified tissues contain hydrographic, nutritional and migrational information--life history information. 4. Utilization of structural and chemical analyses of billfish otoliths, vertebrae, and other hard parts, enables specific phases of growth to be linked to environmental occurrences. Measurement of the metabolically accumulated cellular pigment, lipofuscin, in post-mitotic tissue may make it possible to define physiological age or readiness. Internal and external structural and chemical analyses of otoliths, vertebrae, and other hard parts, combined with biochemical assays of post-mitotic tissue will greatly enhance studies of life history. The methods presented provide insights into means by which life history knowledge can be obtained.     

GROWTH FACTOR INTERACTIONS IN THE TISSUE CULTURE OF TUMOROUS AND NONTUMOROUS NICOTIANA GLAUCA-LANGSDORFFII

Schaeffer , Gideon W., Harold H. Smith and Marion P. Perkus . (Brookhaven Natl. Lab., Upton, N. Y.) Growth factor interactions in the tissue culture of tumorous and nontumorous Nicotiana glauca-langsdorffii. Amer. Jour. Bot. 50(8): 766–771. Illus. 1963.—Tissues representing tumorous and nontumorous Nicotiana glauca-langsdorffii were cultured on high (5 ×) and low (1 ×) concentrations of a modified White's basal medium containing 2.9 × 10^–6m indoleacetic acid. The growth responses of tissues of both the tumorous and nontumorous genotypes to supplements of kinetin, glutamine, inositol and nucleic acid constituents added singly and in all combinations were noted on high-salt media. The nucleic acid components inhibited growth and were omitted from low-salt media. The best growth response was observed with glutamine and inositol for tissues from the tumorous hybrid and with glutamine, inositol and kinetin in the nontumorous type. Kinetin was a distinct and consistent requirement for rapid growth of nontumorous tissues, but no appreciable kinetin effect could be observed with tissues from the tumorous genotype.     

Effect of severity of knee osteoarthritis on the variability of gait parameters

Gait analysis has provided important information concerning gait patterns and variability of gait in patients with knee osteoarthritis (OA) of varying severity. The objective of this study was to clarify how the variability of gait parameters is influenced by the severity of knee OA. Gait analysis was performed at three different controlled walking speeds in three groups of subjects with varying degrees of knee OA (20 healthy subjects with no OA and 90 patients with moderate or severe OA). The variability of gait parameters was characterized by the coefficient of variance (CV) of spatial-temporal parameters, as well as by the mean coefficient variance (MeanCV) of angular parameters. Based on our results, we conclude that the complexity of gait decreases if the walking speed differs from the self-selected speed. In patients with knee OA, the decreased variability of angular parameters on the affected side represents decreased joint flexibility. This leads to decreased consistency in movements of the lower limbs from stride-to-stride, as shown by increased variability of spatial-temporal parameters. Decreased joint flexibility and consistency of movement can be associated with decreased complexity of movement. Other joints of the kinetic chain, such as joints of the non-affected side and the pelvis, play an important role in compensation and adaptation of step-by step motion and in the ability of secure gait. Results suggest that the variability of gait associated with knee osteoarthritis is gender-dependent. During rehabilitation, particular attention must be paid to improving gait stability and proprioception and gender differences should be taken into account.     

Risk of genetic transformation of multipotent mesenchymal stromal cells in vitro

Proof of the efficacy of cell therapy by numerous studies and clinical trials inevitably has raised the question of improving the regulatory framework that governs its use. Particular attention should be paid to the genetic safety of cell preparations. The immune, genetic, and pharmacological modification and expansion of cells in vitro can lead to an undesired effect, which not only has reduced the healing, recovery, and regulatory potential of cell therapy, but also increased the risk of accumulating genetically aberrant cells and the oncogenic transformation of cell preparations. The article has presented the estimation of the parameters of the genetic stability of cultured multipotent mesenchymal stromal cells (MSCs) derived from bone marrow and adipose tissue. The study was conducted using classic methods of genotoxicology, i.e., the individual cells gel-electrophoresis (comet assay) and the micronucleus test. We described a basic level of DNA damage and the frequency of micronucleus, identified genetically instable cultures, and conducted the comparison of genetic variability of MSCs isolated from different tissues.     

REPRODUCTION IN POLEMONIUM: PATTERNS AND IMPLICATIONS OF FLORAL NECTAR PRODUCTION AND STANDING CROPS

Patterns of floral nectar production and standing crop were measured in four populations of the herbaceous perennial plant species Polemonium foliosissimum. Contrary to prediction (Pleasants, 1983), individual flowers in this mass-flowering species were found to produce equivalent nectar volumes every day of their lives. Alternative methods of increasing the reward variability presented to pollinators are evaluated for P. foliosissimum and the relationship between that variability and risk-aversive foraging by pollinators is discussed. Significant spatial and temporal variability in rate of nectar production was found. Populations separated by approximately 200 m exhibited different rates. Nectar production declined significantly as a function of time of the flowering season in two populations but not in a third. In spite of such variability, individual plants showed consistency in production both within a single blooming season and across successive seasons. Because of the variability found in the present study, care should be taken to design appropriate sampling protocols in future nectar studies. Patterns of standing nectar crop were consistent with those expected if pollinators were using an area-restricted searching pattern.     

Survey of the Quality of Experimental Design,Statistical Analysis and Reporting of Research Using Animals

For scientific, ethical and economic reasons, experiments involving animals should be appropriately designed, correctly analysed and transparently reported. This increases the scientific validity of the results, and maximises the knowledge gained from each experiment. A minimum amount of relevant information must be included in scientific publications to ensure that the methods and results of a study can be reviewed, analysed and repeated. Omitting essential information can raise scientific and ethical concerns. We report the findings of a systematic survey of reporting, experimental design and statistical analysis in published biomedical research using laboratory animals. Medline and EMBASE were searched for studies reporting research on live rats, mice and non-human primates carried out in UK and US publicly funded research establishments. Detailed information was collected from 271 publications, about the objective or hypothesis of the study, the number, sex, age and/or weight of animals used, and experimental and statistical methods. Only 59% of the studies stated the hypothesis or objective of the study and the number and characteristics of the animals used. Appropriate and efficient experimental design is a critical component of high-quality science. Most of the papers surveyed did not use randomisation (87%) or blinding (86%), to reduce bias in animal selection and outcome assessment. Only 70% of the publications that used statistical methods described their methods and presented the results with a measure of error or variability. This survey has identified a number of issues that need to be addressed in order to improve experimental design and reporting in publications describing research using animals. Scientific publication is a powerful and important source of information; the authors of scientific publications therefore have a responsibility to describe their methods and results comprehensively, accurately and transparently, and peer reviewers and journal editors share the responsibility to ensure that published studies fulfil these criteria.     

Terrestrial laser scanner based 3D reconstruction of trees and retrieval of leaf area index in a forest environment

Three-dimensional reconstruction of trees and the estimation of biophysical parameters is significant for the management of forest resources, ecological studies carbon cycle and biodiversity. Terrestrial LiDAR data provides detailed, objective and three-dimensional measurement of forest structure and exact metrics of the tree canopies. Several methods for tree detection including canopy height models and raster interpolation models are based on commercial software and huge data processing. The objective of the given study is the three-dimensional reconstruction of trees by implementing segmentation algorithms and thereby estimating the Leaf Area Index of individual tree segments by terrestrial laser scanned data in the Mudumalai forests of Western Ghats, India. The hierarchical minimum cut segmentation method is used for the three-dimensional reconstruction of the individual trees by tracking cylinders along individual branches and trees in a hierarchical order. Super voxel clustering method is also implemented in the study for tree reconstruction and estimating the tree parameters. Leaf area index is calculated by applying a multivariate regression technique for the heights and the diameter obtained from both the segmentation methods. Results obtained indicated a strong correlation with the in-situ measurements which are obtained from the instruments. The approach addresses the applicability of segmentation algorithms which can be run fully automatically. The approach successfully reconstructed a high precision and realistic model of trees in the Western Ghats region which failed in the case of traditional tree modeling methods which requires multiple instruments operating simultaneously for extracting each parameter. The method proved that using TLS; multiple forest parameters can be estimated simultaneously.     

A Bayesian view on cryo-EM structure determination

Three-dimensional (3D) structure determination by single-particle analysis of cryo-electron microscopy (cryo-EM) images requires many parameters to be determined from extremely noisy data. This makes the method prone to overfitting, that is, when structures describe noise rather than signal, in particular near their resolution limit where noise levels are highest. Cryo-EM structures are typically filtered using ad hoc procedures to prevent overfitting, but the tuning of arbitrary parameters may lead to subjectivity in the results. I describe a Bayesian interpretation of cryo-EM structure determination, where smoothness in the reconstructed density is imposed through a Gaussian prior in the Fourier domain. The statistical framework dictates how data and prior knowledge should be combined, so that the optimal 3D linear filter is obtained without the need for arbitrariness and objective resolution estimates may be obtained. Application to experimental data indicates that the statistical approach yields more reliable structures than existing methods and is capable of detecting smaller classes in data sets that contain multiple different structures.     

The efficient physiological strategy of a novel tomato genotype to adapt to chronic combined water and heat stress

• Climate change is increasing the frequency of high temperature shocks and water shortages, pointing to the need to develop novel tolerant varieties and to understand the mechanisms employed to withstand combined abiotic stresses.
• Two tomato genotypes, a heat-tolerant Solanum lycopersicum accession (LA3120) and a novel genotype (E42), previously selected as a stable yielding genotype under high temperatures, were exposed to single and combined water and heat stress. Plant functional traits, pollen viability and physiological (leaf gas exchange and chlorophyll a fluorescence emission measurements) and biochemical (antioxidant content and antioxidant enzyme activity) measurements were carried out. A Reduced Representation Sequencing approach allowed exploration of the genetic variability of both genotypes to identify candidate genes that could regulate stress responses.
• Both abiotic stresses had a severe impact on plant growth parameters and on the reproductive phase of development. Growth parameters and leaf gas exchange measurements revealed that the two genotypes used different physiological strategies to overcome individual and combined stresses, with E42 having a more efficient capacity to utilize the limiting water resources. Activation of antioxidant defence mechanisms seemed to be critical for both genotypes to counteract combined abiotic stresses. Candidate genes were identified that could explain the different physiological responses to stress observed in E42 compared with LA3120.
• Results here obtained have shown how new tomato genetic resources can be a valuable source of traits for adaptation to combined abiotic stresses and should be used in breeding programmes to improve stress tolerance in commercial varieties.

     

Mapping the structure of macromolecular assemblies by combining chemical modification and separation methods

Several examples will be described in which powerful separation methods are combined with relatively simple chemical modification techniques to provide structural information on complex macromolecular assemblies. Ribosomal RNA structure has been examined by crosslinking, separating individual crosslinked species by gel electrophoresis, and enzymatic methods for determination of crosslink positions in the nucleotide sequence. Chromatin structure has been examined by footprinting the location of individual nucleosomes by a combination of chemical nicking and DNA separations. Virus structure can be examined by using breakable crosslinkers analyzed with diagonal gel electrophoresis. Ultimately such methods may allow structural information to be obtained on systems even as complex as whole chromosomes.     

Expression of the differentiation antigen F7D6 in tumorous tissues of Drosophila

The 63-kDa antigen recognized by the monoclonal antibody F7D6 is present in all Drosophila embryonic cells and disappears from most tissues as each one reaches its final, differentiated state. Larval tissues lose the antigen around the time of hatching, imaginal tissues lose it during metamorphosis, and germ cells lose it during gametogenesis (Bedian et al: Devel Biol 115:105-118, 1986). The nervous system and spontaneously contracting musculature of the gut and gonads are exceptions and remain antigen positive at all stages. The F7D6 antigen appears to be associated with dividing, undifferentiated cells and electrogenic cells. This prompted us to test tumors for antigen presence. We tested four different recessive mutants that give rise to four different types of tumorous transformation: the embryonic tumor Notch, several larval melanotic tumors, the imaginal disc tumor 1(2)gl, and three alleles of the ovarian tumor otu. In all cases, tumorous tissues in homozygotes contained the F7D6 antigen. The electrophoretic mobility of the antigen appeared to be unaltered in tumorous tissues compared to normal cells, but the antigen is expressed at higher levels. The antigen is found on the cytoplasmic surface of plasma membranes and appears to be a marker of undifferentiated normal and tumorous cells. Similarities and differences between the F7D6 antigen and Drosophila c-src protein are discussed.     

Mapping physiological states from microarray expression measurements

MOTIVATION: The increasing use of DNA microarrays to probe cell physiology requires methods for visualizing different expression phenotypes and explicitly connecting individual genes to discriminating expression features. Such methods should be robust and maintain biological interpretability. RESULTS: We propose a method for the mapping of the physiological state of cells and tissues from multidimensional expression data such as those obtained with DNA microarrays. The method uses Fisher discriminant analysis to create a linear projection of gene expression measurements that maximizes the separation of different sample classes. Relative to other typical classification methods, this method provides insights into the discriminating characteristics of expression measurements in terms of the contribution of individual genes to the definition of distinct physiological states. This projection method also facilitates visualization of classification results in a reduced dimensional space. Examples from four different cases demonstrate the ability of the method to produce well-separated groups in the projection space and to identify important genes for defining physiological states. The method can be augmented to also include data from the proteomic and metabolic phenotypes and can be useful in disease diagnosis, drug screening and bioprocessing applications.     

Effect of linkage on the control of inbreeding in selection programmes

Selection and mating methods for controlling inbreeding in selection programmes are based on relationships obtained from pedigrees. The efficiency of these methods has always been tested by studies using genetic models of independent loci. However, under linkage the rate of inbreeding obtained from pedigrees can be different from the probability of identity by descent of genes. We simulated a quantitative trait under artificial selection controlled by a large number of genes spread on genome regions of different sizes. A method to control inbreeding based on minimising the average coancestry of selected individuals with a restriction in the loss of selection response, and a mating procedure to control inbreeding were applied. These methods, that use coancestry relationships, were not effective in controlling inbreeding when the genome sizes were smaller than five morgans or so. However, for larger genome sizes the methods were sufficiently efficient. For very tight linkage, methods that utilise molecular information from markers should be used. We finally discuss the effects of the selection of individual major genes on the neutral variability of adjacent genome regions.     

Screening methods for the evaluation of crop allelopathic potential

There is increasing interest in the development of allelopathic crop varieties for weed suppression. Allelopathic varieties are likely to be able to suppress weeds by natural exudation of bioactive allelochemicals, thereby reducing dependence upon synthetic herbicides. Screening bioassays are essential tools in identifying crop accessions with allelopathic potential. A number of crops have been screened for this allelopathic trait, and key issues in selecting and designing screening bioassays are reviewed. It is recommended that a combination of different bioassays be used in the evaluation of crop allelopathic potential. Laboratory bioassays, field testing, and chemical screening are important steps, and none of them can be precluded if conclusive evidence of crop allelopathy is to be established. More concerted efforts are needed in screening crop germplasm before the development of allelopathic varieties occurs.     

Suppression of the Tumorous State in Crown Gall Teratomas of Tobacco A Clonal Analysis

Two types of experimental approaches designed to test the possible involvement of chimeras in the persistent but readily reversible suppression of the tumorous state and in a recovery from that state in crown gall teratomas of tobacco were investigated. These studies showed that the tumorous state was suppressed in specialized cells present in the different tissues derived from each of the three layers of the apical meristem of teratoma-derived shoots of clonal origin. Although differences existed in the exogenous requirements needed for the reestablishment of the tumorous state in the different specialized cell types present in the different tissues, all again acquired a capacity for autonomous growth when appropriately stimulated. No evidence was found for the existence of periclinal or sectoral chimeras in teratoma-derived shoots of a single cell origin. Chimerism was, however, found to exist in one noncloned teratoma-derived shoot. Analysis of more than 500 clones derived from leaf mesophyll cells of cloned teratoma shoots showed that the vast majority of them (> 99%) were tumorous, while all of the 144 seed-grown plants from these shoots had completely recovered from the tumorous state. The results of these studies suggest that (a) a teratoma shoot does not depend for its development on the presence of significant numbers, if any, of cells that had recovered from the tumorous state; (b) the tumorous state may be suppressed in even the most highly specialized cells; (c) some special mechanism exists in the meiotic process for the elimination of all or most of the foreign DNA from transformed cells.     

The ecology and evolution of induced responses to herbivory and how plants perceive risk

1. Plants perceive herbivore damage or increased risk and respond. These changes may increase plant fitness, although effects on fitness have often been assumed without supporting evidence. 2. Three models have been proposed to explain induced rather than constitutive defence. The optimal defence model posits that induction allow plants to reduce allocation costs; it predicts demonstrably lower costs when defences are not needed. The moving target model posits that induction increases spatial and temporal variability; it predicts that variability will be difficult for herbivores and will provide defence. The information transfer model posits that induced responses provide cues to other tissues on that individual plant and to other organisms in the community; it predicts that induced cues will provide systemic resistance, deter herbivores, and/or attract enemies of herbivores, thereby benefiting the induced plant. 3. All three models predict that cues must be reliable to be useful. In some cases, cues provide specific information about the damaged plant tissue and the herbivore and this specific information may allow plants to fine-tune responses. Recent theory posits that selection should favour plants that minimise recognition errors and reduce fitness costs associated with errors. 4. Future research should focus on exploring different modalities used by plants to perceive herbivore risk, the benefits and costs of perceiving cues and inducing resistance, and the basic natural history of these phenomena. Induced responses have great unrealised potential in agriculture, and research should focus on host plant resistance rather than attempting to involve other trophic levels.     

Comparison of four simple methods for estimating sexual dimorphism in fossils

Estimating sexual dimorphism in skeletal and dental features of fossil species is difficult when the sex of individuals cannot be reliably determined. Several different methods of estimating dimorphism in this situation have been suggested: extrapolation from coefficients of variation, division of a sample about the mean or median into two subsamples which are then treated as males and females, and finite mixture analysis (specifically for estimating the maximum dimorphism that could be present in a unimodal distribution). The accuracy of none of these methods has been thoroughly investigated and compared in a controlled manner. Such analysis is necessary because the accuracy of all methods is potentially affected by fluctuations in either sample size, sex ratio, or the magnitude of intrasexual variability. Computer modeling experiments show that the mean method is the least sensitive to fluctuations in these parameters and generally provides the best estimates of dimorphism. However, no method can accurately estimate low to moderate levels of dimorphism, particularly if intrasexual variability is high and sex ratios are skewed. © 1994 Wiley-Liss, Inc.