The use of a hybrid linear trap/FT-ICR mass spectrometer for on-line high resolution/high mass accuracy bottom-up sequencing.

In this work we present a hybrid linear trap/Fourier transform ion cyclotron resonance (ICR) mass spectrometer to perform protein sequencing using the bottom-up approach. We demonstrate that incorporation of the linear trap greatly enhances the overall performance of the hybrid system for the study of complex peptide mixtures separated by fast high-performance liquid chromatography gradients. The ability to detect in the linear trap enables employment of automatic gain control to greatly reduce space charging in the ICR cell irregardless of ion flux. Resulting accurate mass measurements of 2 ppm or better using external calibration are achieved for the base peak as well as ions at 2% relative abundance. The linear trap is used to perform ion accumulation and activation prior to detection in the ICR cell which increases the scan rate. The increased duty cycle allows for data-dependent mass analysis of coeluting peptides to be acquired increasing protein sequence coverage without increasing the gradient length. In addition, the linear trap could be used as an ion detection device to perform simultaneous detection of tandem mass spectra with full scan mass spectral detection in the ICR cell resulting in the fastest scan cycles for performing bottom-up sequencing of protein digests. Comparisons of protein sequence coverage are presented for product ion detection in the linear trap and ICR cell.     

Genetic variation in two populations of the rough‐skinned newt (Taricha granulosa) assessed using novel tetranucleotide microsatellite loci

Seven novel tetranucleotide microsatellite loci were identified from a partial genomic DNA library, enriched for GATA‐motif microsatellites, from the rough‐skinned newt (Taricha granulosa). All loci were polymorphic, and one displayed a high frequency null allele. A related species, T. rivularis, displays strong site fidelity and detectable population structure over small spatial scales, so we assessed genetic variation in two samples of T. granulosa separated by 16 km. Distributions of allele frequencies differ significantly between our two sites, but small values of F_ST and Rho_ST suggest that the populations are linked by a large amount of gene flow.     

Non-native and translocated fish species in Serbia and their impact on the native ichthyofauna

A total of 22 fish species have been introduced into the inland waters of Serbia, either intentionally or accidentally. This paper provides a summary of data concerning time and reason of introduction, mode of expansion, degree of acclimatization, impact on native fish and estimated area of recent distribution. Four of the non-native fish species currently occupy more than 51% of Serbian territory while 5 of them occupy between 21–50% of territory. This paper reviews impacts of introduced freshwater fish in Serbia based on collected data.     

A note on the sensitivity of the histochemical plasmal reaction

Summary Comparison of the results from both histochemical and biochemical determinations of the plasmalogen concentration in several tissues and organs shows that the two methods are in general agreement. A comparison of the two methods also indicates that the lowest concentration of plasmalogen detectable with the histochemical procedure is about 0.7 moles per gram when 10 sections are employed.This investigation was supported by USPHS Grant 5TI-GM-407.A portion of this paper was prepared from data in a dissertation submitted to the Faculty of the Graduate School of the State University of New York at Buffalo, in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Ontogenetic plasticity of anatomical and ecophysiological traits and their correlations in Iris pumila plants grown in contrasting light conditions

To better understand what directs and limits the evolution of phenotype, constraints in the realization of the optimal phenotype need to be addressed. That includes estimations of variability of adaptively important traits as well as their correlation structures, but also evaluation of how they are affected by relevant environmental conditions and development phases. The aims of this study were to analyze phenotypic plasticity, genetic variability and correlation structures of important Iris pumila leaf traits in different light environments and ontogenetic phases, and estimate its evolutionary potential. Stomatal density, specific leaf area, total chlorophyll concentration and chlorophyll a/b ratio were analyzed on I. pumila full‐sib families in the seedling phase and on the same plants after 3 years of growth in contrasting light conditions typical for ontogenetic stage in question. There was a significant phenotypic plasticity in both ontogenetic stages, but significant genetic variability was detected only for chlorophyll concentrations. Correlations of the same trait between different stages were weak due to changes in environmental conditions and difference in ontogenetic reaction norms of different genotypes. Ontogenetic variability of correlation structures was detected, where correlations and integration were higher in seedlings compared with adult plants 3 years later. Correlations were affected by environmental conditions, with integration being higher in the lower light conditions, but correlations between phases being stronger in the higher light treatment. These findings demonstrated that the analyzed traits can be selected and can mostly evolve independently in different environments and ontogenetic stages, with low genetic variability as a potentially main constraint.     

Regulation of the subcellular localization of cyclic AMP-dependent protein kinase in response to physiological stresses and sexual differentiation in the fission yeast Schizosaccharomyces pombe

We describe regulation of the subcellular localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) regulatory (Cgs1p) and catalytic (Pka1p) subunits in the fission yeast Schizosaccharomyces pombe in response to physiological stresses and during sexual differentiation as determined by fluorescence microscopy of the Cgs1-green fluorescent protein (GFP) and Pka1-GFP fusion proteins, respectively. In wild-type S. pombe cells cultured to log phase under normal growth conditions, Cgs1p and Pka1p are concentrated in the nucleus and more diffusely present in the cytoplasm. Nuclear localization of both proteins is dependent on cAMP, since in cells lacking adenylate cyclase they are detectable only in the cytoplasm. In cells lacking Cgs1p or both Cgs1p and adenylate cyclase, Pka1p is concentrated in the nucleus, demonstrating a role for Cgs1p in the nuclear exclusion of Pka1p. Nuclear-cytoplasmic redistribution of Cgs1p and Pka1p is triggered by growth in glucose-limited or hyperosmotic media and in response to stationary-phase growth. In addition, both proteins are excluded from the nucleus in mating cells undergoing karyogamy and subsequently concentrated in postmeiotic spores. Cgs1p is required for subcellular redistribution of Pka1p induced by growth in glucose-limited and hyperosmotic media and during karyogamy but is not required for Pka1p redistribution triggered by stationary-phase growth or for the enrichment of Pka1p in spores. Our results demonstrate that PKA localization is regulated by cAMP and regulatory subunit-dependent and -independent mechanisms in S. pombe.     

Hypoxia and stem cell‐based engineering of mesenchymal tissues

Stem cells have the ability for prolonged self‐renewal and differentiation into mature cells of various lineages, which makes them important cell sources for tissue engineering applications. Their remarkable ability to replenish and differentiate in vivo is regulated by both intrinsic and extrinsic cellular mechanisms. The anatomical location where the stem cells reside, known as the “stem cell niche or microenvironment,” provides signals conducive to the maintenance of definitive stem cell properties. Physiological condition including oxygen tension is an important component of the stem cell microenvironment and has been shown to play a role in regulating both embryonic and adult stem cells. This review focuses on oxygen as a signaling molecule and the way it regulates the stem cells' development into mesenchymal tissues in vitro. The physiological relevance of low oxygen tension as an environmental parameter that uniquely benefits stem cells' expansion and maintenance is described along with recent findings on the regulatory effects of oxygen on embryonic stem cells and adult mesenchymal stem cells. The relevance to tissue engineering is discussed in the context of the need to specifically regulate the oxygen content in the cellular microenvironment in order to optimize in vitro tissue development. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Coevolution of Interacting Fertilization Proteins

Reproductive proteins are among the fastest evolving in the proteome, often due to the consequences of positive selection, and their rapid evolution is frequently attributed to a coevolutionary process between interacting female and male proteins. Such a process could leave characteristic signatures at coevolving genes. One signature of coevolution, predicted by sexual selection theory, is an association of alleles between the two genes. Another predicted signature is a correlation of evolutionary rates during divergence due to compensatory evolution. We studied female–male coevolution in the abalone by resequencing sperm lysin and its interacting egg coat protein, VERL, in populations of two species. As predicted, we found intergenic linkage disequilibrium between lysin and VERL, despite our demonstration that they are not physically linked. This finding supports a central prediction of sexual selection using actual genotypes, that of an association between a male trait and its female preference locus. We also created a novel likelihood method to show that lysin and VERL have experienced correlated rates of evolution. These two signatures of coevolution can provide statistical rigor to hypotheses of coevolution and could be exploited for identifying coevolving proteins a priori. We also present polymorphism-based evidence for positive selection and implicate recent selective events at the specific structural regions of lysin and VERL responsible for their species-specific interaction. Finally, we observed deep subdivision between VERL alleles in one species, which matches a theoretical prediction of sexual conflict. Thus, abalone fertilization proteins illustrate how coevolution can lead to reproductive barriers and potentially drive speciation.     

Crystal structure of the agrin-responsive immunoglobulin-like domains 1 and 2 of the receptor tyrosine kinase MuSK

Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed exclusively in skeletal muscle, where it is required for formation of the neuromuscular junction. MuSK is activated by agrin, a neuron-derived heparan sulfate proteoglycan. Here, we report the crystal structure of the agrin-responsive first and second immunoglobulin-like domains (Ig1 and Ig2) of the MuSK ectodomain at 2.2 A resolution. The structure reveals that MuSK Ig1 and Ig2 are Ig-like domains of the I-set subfamily, which are configured in a linear, semi-rigid arrangement. In addition to the canonical internal disulfide bridge, Ig1 contains a second, solvent-exposed disulfide bridge, which our biochemical data indicate is critical for proper folding of Ig1 and processing of MuSK. Two Ig1-2 molecules form a non-crystallographic dimer that is mediated by a unique hydrophobic patch on the surface of Ig1. Biochemical analyses of MuSK mutants introduced into MuSK(-/-) myotubes demonstrate that residues in this hydrophobic patch are critical for agrin-induced MuSK activation.