Protein identification by liquid chromatography-mass spectrometry using retention time prediction

Liquid chromatography has been coupled with mass spectrometry to improve the dynamic range and to reduce the complexity of sample introduced to the mass spectrometer at any given time. The chromatographic separation also provides information on the analytes, such as peptides in enzymatic digests of proteins; information that can be used when identifying the proteins by peptide mass fingerprinting. This paper discusses a recently introduced method based on retention time prediction to extract information from chromatographic separations and the applications of this method to protein identification in organisms with small and large genomes.     

Spatial heterogeneity recognition in estuarine intertidal benthic macrofaunal communities: influence of sieve mesh-size and sampling depth

Estuarine intertidal soft-bottom macrobenthic infauna of the Tagus estuary was characterised using different mesh size sieves and sediment sampling depth. The study sampled 105 sites using a hand held 0.01 m² corer. The top layer (0–5 cm) was sieved through nested 1.0 and 0.5 mm meshes whereas the bottom layer (5–20 cm) was through a 1 mm mesh. The total survey took 26 taxa of more than 5800 individuals and a total wet weight biomass of over 650 g. The top layer, using both sieves, gathered 23 taxa (92% of the total), more than 5600 specimens (96%) but less than 8 g of biomass (1%) whereas the 1.0 mm sieve retained 21 taxa (91%), more than 1700 specimens (31%) and almost 7 g of biomass (1%). Abundance was dominated by small annelids, of which Streblospio shrubsolii was 68%, whereas biomass was dominated by molluscs, with the bivalve Scrobicularia plana representing 98%. Multivariate analyses showed an abundance pattern where the top layer data was very similar to that obtained with both layers. The bottom layer data were needed to accurately represent the total biomass pattern. The macrofaunal spatial pattern identified with the 0.5 mm sieve data differed from that identified by the 1.0 mm and was essential to discriminate a faunal assemblage located along the upper part of the shore. It was concluded that in order to characterize the macrofauna community structure, based on the presence/absence of taxa, the top layer and a 1.0 mm sieve would be sufficient. An abundance-based characterization requires the top layer and a 0.5 mm sieve whereas a biomass-based characterization requires data for both layers but it is sufficient to use the 1.0 mm sieve. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Living sieve cells of conifers as visualized by confocal,laser-scanning fluorescence microscopy

Summary Confocal laser scanning microscopy (CLSM) and fluorochromes were used to visualize the assimilate-conducting sieve cells of conifers in vivo. When still nucleate, the cytoplasm of these cells shows streaming and occupies the cell periphery including the pitlike, thin wall regions where sieve areas would develop. During differentiation the nuclear fluorescence and the central vacuoles disappear. At maturity and after ER-specific staining the sieve areas are the most conspicuous character of sieve cells. Those linking two sieve cells are covered on either side with prominent amounts of ER, while those leading to a Strasburger (=albuminous) cell show fluorescence on the sieve-cell side only. Within the sieve-area wall fluorescence appears also in the common median cavity which is part of the symplastic path between sieve cells. Electron microscopy (EM) depicts the ER as complexes of densely convoluted tubules of smooth ER, equally on either side of a sieve area, provided that the fixation of this sensitive tissue is appropriate. Purposeful wounding causes a swelling and vesiculation of the ER-tubules which is visible in both CLSM and EM. Electron micrographs of ER-complexes at sieve areas -in this paper demonstrated in vivo -have often been argued to be artefacts, since they should raise flow resistance considerably and are not consistent with the Münch hypothesis on phloem transport. The implications of this location for phloem transport are discussed.Abbreviations CLSM confocal laser scanning microscopy - DiOC 3,3-dioxacarbocyanine iodide - EM electron microscopy - ER endoplasmic reticulum - FDA fluorescein diacetate     

Evaluating the importance of predation on subtidal benthic assemblages in sandy habitats around rocky reefs

It was proposed that predation could explain differences in the structure of the benthic macrofaunal assemblages in subtidal sandy sediments close to compared with those far from rocky reefs. This hypothesis was tested using experimental exclusion cages and partial cages at two sites at two distances at two different rocky reefs. Undisturbed uncaged assemblages of macrofauna close to the rocky reefs were generally different from those in the partial cages and full cages. However, caging artifacts could not be detected and there were no strong correlations between the macrofauna and the proportions of different grain size and organic content. The structure of the macrofaunal assemblages close to the rocky reefs was, nevertheless, different from those far from the reefs and the sediments were finer far from than close to the rocky reefs. The results indicated that factors other than predation or grain size caused the differences in the macrofauna. For the spatial and temporal scales used in this study, it was clear that, although predation maybe intense, on its own it cannot explain the differences in the structure of the assemblages close and far from rocky reefs. The importance of adequate replication on caging experiments is discussed and it is suggested that alternative ways need to be found to test predictions about the influence of predation on soft sediment benthic assemblages.     

Reducing sample complexity of polyclonal human autoantibodies by chromatofocusing

Chromatofocusing was performed in order to separate a polyclonal antigen-specific mixture of human immunoglobulins (IgGs) that would then allow for further analyses of as few different IgGs as possible. Because polyclonal IgGs only differ by amino acid sequence and possible post-translational modifications but not by molecular weight, we chose chromatofocusing for protein separation by different isoelectric points. We isolated antigen-specific IgGs from commercially available intravenous immunoglobulins (IVIG) using a combination of affinity- and size exclusion-chromatography and in order to reduce the complexity of the starting material IVIG was then replaced by single-donor plasmapheresis material. Using two-dimensional gel electrophoresis (2-DE), we observed a clear decrease in the number of different light and heavy chains in the chromatofocusing peak as compared to the starting material. In parallel, we monitored slight problems with the selected peak in isoelectric focusing as the first dimension of 2-DE, displayed in by the less proper focusing of the spots. When we tested whether IgGs were binding to their specific antigen after chromatofocusing, we were able to show that they were still in native conformation. In conclusion, we showed that chromatofocusing can be used as a first step in the analysis of mixtures of very similar proteins, e.g. polyclonal IgG preparations, in order to minimize the amount of different proteins in separated fractions in a reproducible way.     

Proliferating sieve elements present in bud phloem anastomoses connect sieve tubes of axillary bud traces to stelar vascular bundles in the aquatic monocotyledonPotamogeton natans L. (Potamogetonaceae)

Summary The stem ofPotamogeton natans is characterized by a central stelar vascular system with reduced xylem and abundant phloem. Wide sieve tubes composed of short sieve-tube members joined by simple sieve plates and associated with companion cells establish an effective conduit for assimilates. At each node the phloem forms a network of parallel sieve elements connecting the stem phloem to leaf and bud traces. InP. natans an axillary bud rarely develops into a side branch, its procambial vascular bundles are each connected to the nodal complex via separate anastomoses. Their most unusual components are the anastomosai sieve elements (ANSE), characterized by thin cell walls pitted all over by tiny callose-lined pores resembling plasmodesmata, which can be detected as bright areas by fluorescence microscopy after staining with aniline blue. Several layers of ANSE make up the centre of an anastomosis and link to both the nodal and bud stelar sieve tubes via mediating (MSE) and connecting sieve elements (CSE). The ultrastructural differentiation of ANSE, MSE, and CSE corresponds to that of normal sieve elements, i.e., in the mature stage they are enucleate, evacuolate, and have lost most of their cytoplasm. Their plastids are of form-P2c, containing many cuneate protein crystals, typical of monocotyledonous sieve elements. Quantitative aspects of the pore areas are discussed in relation to the functional significance of bud anastomoses.Abbreviations ANSE anastomosai sieve elements - CSE connecting sieve elements - FM fluorescence microscopy - LM light microscopy - MSE mediating sieve elements - TEM transmission electron microscopy Dedicated to Professor Dr. Rainer Kollmann on the occasion of his retirement     

Longevity cost of reproduction for males but no longevity cost of mating or courtship for females in the male-dimorphic dung beetle Onthophagus binodis

Life history theory predicts a trade-off between current and future reproduction. Despite a wealth of research on the cost of reproduction for females, there have been very few studies that have looked at the cost of reproduction for males. Longevity is closely related to the opportunity for future reproduction, and thus decreased longevity in response to current reproductive effort has been used as a measure of the cost of reproduction. Here we examine the cost of reproduction for males and females in the dung beetle Onthophagus binodis. Like many onthophagines, O. binodis exhibit dimorphic male morphology; major males develop a large pronotal horn while minor males remain hornless. Alternative morphologies are associated with alternative reproductive tactics. Thus, we ask whether major and minor males pay different costs of reproduction. We found that in contrast to previous work on Diptera, mating is not costly in terms of reduced longevity for female dung beetles. Despite a longevity cost of reproduction for males, we found no evidence for differential longevity costs associated with alternative reproductive tactics.     

Mathematical optimization of procedures for cryoprotectant equilibration using a toxicity cost function

Cryopreservation nearly universally depends on the equilibration of cells and tissues with high concentrations of permeating chemicals known as cryoprotective agents, or CPAs. Despite their protective properties, CPAs can cause damage as a result of osmotically-driven cell volume changes, as well as chemical toxicity. In this study, we have used previously published data to determine a toxicity cost function, a quantity that represents the cumulative damage caused by toxicity. We then used this cost function to define and numerically solve the optimal control problem for CPA equilibration, using human oocytes as representative cell type with high clinical relevance. The resulting toxicity-optimal procedures are predicted to yield significantly less toxicity than conventional stepwise procedures. In particular, our results show that toxicity is minimized during CPA addition by inducing the cell to swell to its maximum tolerable volume and then loading it with CPA while in the swollen state. This counterintuitive result is considerably different from the conventional stepwise strategy, which involves exposure to successively higher CPA concentrations in order to avoid excessive shrinkage. The procedures identified in the present study have the potential to significantly reduce damage due to toxicity and warrant further investigation.     

An evaluation of the analysis system of video transects used to sample subtidal epibiota

Two popular methods of benthic cover estimation (the point intercept technique with two sets of position points, and digital interactive color segmentation) were compared with an alternative method of digital cover estimation using Bezier curves as a tool for outlining the objects on an images and AutoCAD® software for the final evaluation of abundance. The comparison was done using still video images obtained from two 10-m transects on subtidal rocks off the Rimsky-Korsakov islands in the Sea of Japan (Russia). Ten rectangular sectors (1×0.4 m each) selected randomly within both video transects were analyzed. One-way ANOVA for repeated measurements was used to test the differences between methods. The point intercept technique differed significantly from both methods of digital estimation and had an essential positive bias. The Tukey multiple comparison test revealed the differences among digital estimation methods in the species, which have the complicated color with many contrast spots. The proposed approach using Bezier curves has an advantage over the interactive color segmentation if the objects under selection are lit at different levels or have a contrast coloration or are hidden by canopy organisms. Besides, estimation of cover in the field of AutoCAD® software is more precise and takes less time than that obtained using a scaled grid, available for automatically segmented species. The results showed that digital cover estimation using Bezier curves and AutoCAD® software is a convenient method for analyzing benthic samples at large spatial scales.     

Optimization of processing conditions for the pretreatment of wheat straw using aqueous ionic liquid

Pretreatment of wheat straw with the aqueous ionic liquid, 1-ethyl-3-methylimidazolium acetate, was optimized to maximize fermentable sugars recovery. The optimization process employed a central composite design, where the investigated variables were temperature (130-170 °C), time (0.5-5.5 h) and ionic liquid concentration (0-100%). All the tested variables were identified to have significant effects (p < 0.05) on fermentable sugars recovery. The optimum pretreatment conditions were 158 °C, an ionic liquid concentration of 49.5% (w/w), and a duration of 3.6 h. Cellulose and xylan digestibility generally increased with increasing temperature, time and ionic liquid concentration; but, the carbohydrates recovered in the washed solids following pretreatment decreased. Thus, the final optimum conditions for maximizing fermentable sugars from the starting biomass were a compromise between greater digestibility and minimal carbohydrates loss during pretreatment.     

Immunolocalization of plasma-membrane H+-ATPase and tonoplast-type pyrophosphatase in the plasma membrane of the sieve element-companion cell complex in the stem of Ricinus communis L

Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L. and, for comparison, in stems of Cucurbita pepo L. and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue. The plasma-membrane (PM) H⁺-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies. Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM. Different PM H⁺-ATPase antibody clones, raised against the PM H⁺-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types. Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H⁺-ATPase isoforms. The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K⁺ and amino acids, as one of the unknown roles of ATP found in SEs. The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes. Received: 3 July 2000 / Accepted: 12 October 2000     

A long-term comparison of the benthic algal flora of Clare Island, County Mayo, western Ireland

The marine benthic algal flora of Clare Island, off County Mayo, western shore of Ireland, was investigated; collections of intertidal and subtidal marine algae were made at 16 sites along the eastern and southern shores in the years 1990, 1993 and 2000–2002. The data and observations obtained were compared with the results of a similar survey conducted by Arthur Disbrowe Cotton in 1910–1911. Considering the results of the original survey and the new survey together, the marine algal flora of the island currently totals 293 species; 224 species were recorded by Cotton in the original survey, whereas 223 species were identified in the present study. Most species are common to the original and the new list and the main differences are easily explainable; the new survey used SCUBA diving, which allowed the collection of several subtidal species not collected in 1910, and Cotton reported several microscopic green and brown algae, usually difficult to recognise in the field, which were not rediscovered. The most remarkable differences consist in the current presence of some large intertidal brown algae (Bifurcaria bifurcata, Cystoseira foeniculacea and Cystoseira nodicaulis) that were not reported in the survey of 1910. Two algae, Codium fragile subsp. tomentosoides and Asparagopsis armata, were introduced in Europe after the original survey. At present, the benthic algal assemblages of Clare Island still have basically the same structure and distribution as in 1910 and, if compared with other coastal areas of Europe, the intertidal marine environment of Clare Island appears remarkably well conserved.     

The cost of reproduction in the glaucous-winged gull

Summary Experimental enlargement of brood size in the glaucous-winged gull (Larus glaucescens) resulted in increased adult foraging time, decreased adult body weight at the end of the breeding season, and decreased over-winter adult survival. The decreased survival of breeding adults was associated with reduced body condition at the end of breeding (resulting from physiological costs of reproduction). Decreased survival was not due to an increased risk of injury or predation during the breeding season. Brood size did not directly affect the fecundity of surviving birds in the subsequent year. However, brood size may have an indirect effect on subsequent fecundity because the probability of mate loss increased among birds with large broods and the reproductive performance of birds with new mates was reduced. Based on estimates of life-time fitness calculated from fecundity and survivorship, birds with two- or three-chick broods (the normal brood size) have higher fitness than birds with one- or four-chick broods. However, the decreased fitness of birds with four-chick broods was slight, and probably not a sufficient explanation for the absence of natural four-chick broods in the glaucouswinged gull.     

Symplastic isolation of the sieve element-companion cell complex in the phloem of Ricinus communis and Salix alba stems

The anatomical and physiological isolation of the sieve element-companion cell complex (se-cc complex) was investigated in stems of Ricinus communis L. and Salix alba L. In Ricinus, the plasmodesmatal frequencies were in the proportions 8∶1∶2∶30, in the order given, at the interfaces between sieve tube-companion cell, sieve tube-phloem parenchyma cell, companion cellphloem parenchyma cell, and phloem parenchyma cellphloem parenchyma cell. The membrane potentials of the se-cc complex and the surrounding phloem-parenchyma cells sharply contrasted: the membrane potential of the se-cc complex was about twice as negative as that of the phloem parenchyma. Lucifer Yellow CH injected into the sieve element or into the companion cell remained within the se-cc complex. Dye introduced into phloem parenchyma only moved (mostly poorly) to other phloem-parenchyma cells. The distribution of the plasmodesmatal frequencies, the differential dye-coupling and the sharp discontinuities in membrane potentials indicate that the se-cc complexes constitute symplast domains in the stem phloem. Symplastic autonomy is discussed as a basic necessity for the functioning of the se-cc complex in the stem.     

Microelectrode-recorded development of the symplasmic autonomy of the sieve element/companion cell complex in the stem phloem of Lupinus luteus L.

From the cambial stage onwards, the symplasmic autonomy of sieve element/companion cell complexes (SE/CC-complexes) was followed in stems of Lupinus luteus L. by microinjection techniques. The membrane potential and the symplasmic autonomy of the mature SE/CC-complex was measured in successive internodes. A microelectrode was inserted into SE/CC-complexes or phloem parenchyma cells (PPs) and, after stabilization of the membrane potential, the membrane-impermeant fluorescent dye Lucifer Yellow CH (LYCH) was injected intracellullary. The plasmodesmata of the cambial SE/ CC precursor were gradually shut off at all interfaces beginning at the walls to be transformed into sieve plates. In the course of maturation, symplasmic discontinuity was maintained at the longitudinal walls of the complex. In the transverse walls of the SE, wide sieve pores were formed giving rise to longitudinal multicellular symplasmic domains of SE/CC-complexes. Symplasmic isolation of the files of mature SE/CC-complexes was demonstrated in several ways: (i) the membrane potential of the SE/CC-complexes (between -100 mV and -130 mV) was consistently more negative than that of the PPs (between-50 and -100 mV), (ii) No exchange of LYCH was observed between SE/CC-complexes and the PPs. Lucifer Yellow CH injected into the SEs exclusively moved to the associated CCs and to other SE/CC-complexes whereas LYCH injected into the PPs was only displaced to other PPs. (iii) The electrical coupling ratio between adjacent PPs was ten times higher than that between SE/CC-complex and PP. A gradient in the membrane potential of the SE/CC-complexes along the stem was not conclusively demonstrated.Abbreviations LYCH Lucifer Yellow CH - membrane potential - PMF proton-motive force - PP phloem parenchyma cell - SE/CC-complex sieve element/companion cell complex - SR-G sulphorhodamine G     

An analytical estimation of the energy cost for legged locomotion

Legged locomotion requires the determination of a number of parameters such as stride period, stride length, order of leg movements, leg trajectory, etc. How are these parameters determined? It has been reported that the locomotor patterns of many legged animals exhibit common characteristics, which suggests that there exists a basic strategy for legged locomotion. In this study we derive an equation to estimate the cost of transport for legged locomotion and examine a criterion of the minimization of the transport cost as a candidate of the strategy. The obtained optimal locomotor pattern that minimizes the cost suitably represents many characteristics of the pattern observed in legged animals. This suggests that the locomotor pattern of legged animals is well optimized with regard to the energetic cost. The result also suggests that the existence of specific gait patterns and the phase transition between them could be the result due to optimization; they are induced by the change in the distribution of ground reaction forces for each leg during locomotion.     

Ultrastructural features of well-preserved and injured sieve elements: Minute clamps keep the phloem transport conduits free for mass flow

Summary After chemical fixation following two different preparation procedures, the ultrastructure of mature sieve elements (SEs) was systematically compared in the transport phloem ofVicia faba leaves andLycopersicon esculentum internodes. The SEs in samples obtained by gentle preparation were well preserved, while those in conventionally prepared samples were generally injured. (1) In well-preserved SEs, parietal P-proteins were associated with cisternae of the SE endoplasmic reticulum (ER). Additionally, theV. faba SEs had crystalline P-proteins, and a homogeneous network of filamentous P-proteins occurred in the lumen of theL. esculentum SEs. In injured SEs, all P-proteins were dispersed. (2) In well-preserved SEs, stacked ER cisternae associated with P-proteins lay also on the sieve-plate walls, but passages were kept free in front of the sieve pores. Injured SEs lacked these orderly arranged deposits. Instead, irregular filamentous and membranous materials occluded the sieve pores. (3) In well-preserved SEs, the sieve-pore lumen was free of obstructions, apart from small, lateral coatings of P-proteins. Sieve pores in injured SEs were always occluded. (4) The SE organelles and, in tomato SEs, also the parietal ER located at the longitudinal walls were firmly attached in the SE periphery and stayed in place after injury. The stable parietal attachment is likely exerted by minute, clamplike structures which link the outer membranes of the SE components with one another or to the SE plasma membrane. Single, straight clamps with a length of about 7 nm anchored the SE components directly to the SE plasma membrane. The connections between adjacent SE organelles and/or parietal ER cisternae were mostly twice as long (about 15 nm) and often were branched. Presumably, the long, branched clamps were constituted by the interaction of opposite short clamps. The ultrastructural results are discussed with respect to SE functioning.     

Reducing the allowable kinetic space by constructing ensemble of dynamic models with the same steady-state flux

Dynamic models of metabolism are instrumental for gaining insight and predicting possible outcomes of perturbations. Current approaches start from the selection of lumped enzyme kinetics and determine the parameters within a large parametric space. However, kinetic parameters are often unknown and obtaining these parameters requires detailed characterization of enzyme kinetics. In many cases, only steady-state fluxes are measured or estimated, but these data have not been utilized to construct dynamic models. Here, we extend the previously developed Ensemble Modeling methodology by allowing various kinetic rate expressions and employing a more efficient solution method for steady states. We show that anchoring the dynamic models to the same flux reduces the allowable parameter space significantly such that sampling of high dimensional kinetic parameters becomes meaningful. The methodology enables examination of the properties of the model's structure, including multiple steady states. Screening of models based on limited steady-state fluxes or metabolite profiles reduces the parameter space further and the remaining models become increasingly predictive. We use both succinate overproduction and central carbon metabolism in Escherichia coli as examples to demonstrate these results.