Agrobacterium-mediated transformation and assessment of factors influ-encing transgene expression in loblolly pine (Pinus taeda L.)

INTRODUCTIONRecombinant DNA technology is a powerful toolfor the introduction of foreign genes into longlivedperennials and fOr fundamelltal studies of gene expression. Using such techniques, we can overcomethe difficulties associated with the breeding of a long-lived perennial. At present, although considerablereseaxch effort has been devoted to the genetic en-gineering of fOrest trees, it has lagged behind ad-vances made in herbaceous crops due both to eco-nomics and the recalcitrant n…     

A novel method reveals that solvent water favors polyproline II over beta-strand conformation in peptides and unfolded proteins: conditional hydrophobic accessible surface area (CHASA)

In aqueous solution, the ensemble of conformations sampled by peptides and unfolded proteins is largely determined by their interaction with water. It has been a long-standing goal to capture these solute-water energetics accurately and efficiently in calculations. Historically, accessible surface area (ASA) has been used to estimate these energies, but this method breaks down when applied to amphipathic peptides and proteins. Here we introduce a novel method in which hydrophobic ASA is determined after first positioning water oxygens in hydrogen-bonded orientations proximate to all accessible peptide/protein backbone N and O atoms. This conditional hydrophobic accessible surface area is termed CHASA. The CHASA method was validated by predicting the polyproline-II (P(II)) and beta-strand conformational preferences of non-proline residues in the coil library (i.e., non-alpha-helix, non-beta-strand, non-beta-turn library derived from X-ray elucidated structures). Further, the method successfully rationalizes the previously unexplained solvation energies in polyalanyl peptides and compares favorably with published experimentally determined P(II) residue propensities. We dedicate this paper to Frederic M. Richards.     

Are proteins made from a limited parts list?

Understanding the process of protein folding has been recognized as an important challenge for >70 years. It is, quintessentially, a thermodynamic problem and, arguably, thermodynamics is our most powerful discipline for understanding biological systems. Yet, despite all this, we still lack predictive understanding of protein folding. Is something missing from this picture?     

A likelihood method for the detection of selection and recombination using nucleotide sequences

Different regions along nucleotide sequences are often subject to different evolutionary forces. Recombination will result in regions having different evolutionary histories, while selection can cause regions to evolve at different rates. This paper presents a statistical method based on likelihood for detecting such processes by identifying the regions which do not fit with a single phylogenetic topology and nucleotide substitution process along the entire sequence. Subsequent reanalysis of these anomalous regions may then be possible. The method is tested using simulations, and its application is demonstrated using the primate psi eta-globin pseudogene, the V3 region of the envelope gene of HIV-1, and argF sequences from Neisseria bacteria. Reanalysis of anomalous regions is shown to reveal possible immune selection in HIV-1 and recombination in Neisseria. A computer program which implements the method is available.      

AdcAII of Streptococcus pneumoniae Affects Pneumococcal Invasiveness

Across bacterial species, metal binding proteins can serve functions in pathogenesis in addition to regulating metal homeostasis. We have compared and contrasted the activities of zinc (Zn²⁺)-binding lipoproteins AdcA and AdcAII in the Streptococcus pneumoniae TIGR4 background. Exposure to Zn²⁺-limiting conditions resulted in delayed growth in a strain lacking AdcAII (ΔAdcAII) when compared to wild type bacteria or a mutant lacking AdcA (ΔAdcA). AdcAII failed to interact with the extracellular matrix protein laminin despite homology to laminin-binding proteins of related streptococci. Deletion of AdcA or AdcAII led to significantly increased invasion of A549 human lung epithelial cells and a trend toward increased invasion in vivo. Loss of AdcAII, but not AdcA, was shown to negatively impact early colonization of the nasopharynx. Our findings suggest that expression of AdcAII affects invasiveness of S. pneumoniae in response to available Zn²⁺ concentrations.     

Detection of microspheres in vivo using multispectral optoacoustic tomography

We introduce a new approach to detect individual microparticles that contain NIR fluorescent dye by multispectral optoacoustic tomography in the context of the hemoglobin-rich environment within murine liver. We encapsulated a near infrared (NIR) fluorescent dye within polystyrene microspheres, then injected them into the ileocolic vein, which drains to the liver. NIR absorption was determined using multispectral optoacoustic tomography. To quantitate the minimum diameter of microspheres, we used both colorimetric and spatial information to segment the regions in which the microspheres appear. Regional diameter was estimated by doubling the maximum regional distance. We found that the minimum microsphere size threshold for detection by multispectral optoacoustic tomography images is 78.9 µm.     

SNL fibroblast feeder layers support derivation and maintenance of human induced pluripotent stem cells

Induced pluripotent stem(iPS) cells can be derived from human somatic cells by cellular reprogramming.This technology provides a potential source of non-controversial therapeutic cells for tissue repair,drug discovery,and opportunities for studying the molecular basis of human disease.Normally,mouse embryonic fibroblasts(MEFs) are used as feeder layers in the initial derivation of iPS lines.The purpose of this study was to determine whether SNL fibroblasts can be used to support the growth of human iPS cell...     

Measuring rapid hydrogen exchange in the homodimeric 36 kDa HIV-1 integrase catalytic core domain

Measurements of rapid hydrogen exchange (HX) of water with protein amide sites contain valuable information on protein structure and function, but current NMR methods for measuring HX rates are limited in their applicability to large protein systems. An alternate method for measuring rapid HX is presented that is well-suited for larger proteins, and we apply the method to the deuterated, homodimeric 36 kDa HIV-1 integrase catalytic core domain (CCD). Using long mixing times for water-amide magnetization exchange at multiple pH values, HX rates spanning more than four orders of magnitude were measured, as well as NOE cross-relaxation rates to nearby exchangeable protons. HX protection factors for the CCD are found to be large (>10(4)) for residues along the dimer interface, but much smaller in many other regions. Notably, the catalytic helix (residues 152-167) exhibits low HX protection at both ends, indicative of fraying at both termini as opposed to just the N-terminal end, as originally thought. Residues in the LEDGF/p75 binding pocket also show marginal stability, with protection factors in the 10-100 range (～1.4-2.7 kcal/mol). Additionally, elevated NOE cross-relaxation rates are identified and, as expected, correspond to proximity of the amide proton to a rapidly exchanging proton, typically from an OH side chain. Indirect NOE transfer between H(2) O and the amide proton of I141, a residue in the partially disordered active site of the enzyme, suggests its proximity to the side chain of S147, an interaction seen in the DNA-bound form for a homologous integrase.     

The Protein Coil Library: a structural database of nonhelix, nonstrand fragments derived from the PDB

Approximately half the structure of folded proteins is either alpha-helix or beta-strand. We have developed a convenient repository of all remaining structure after these two regular secondary structure elements are removed. The Protein Coil Library (http://roselab.jhu.edu/coil/) allows rapid and comprehensive access to non-alpha-helix and non-beta-strand fragments contained in the Protein Data Bank (PDB). The library contains both sequence and structure information together with calculated torsion angles for both the backbone and side chains. Several search options are implemented, including a query function that uses output from popular PDB-culling servers directly. Additionally, several popular searches are stored and updated for immediate access. The library is a useful tool for exploring conformational propensities, turn motifs, and a recent model of the unfolded state.     

Steric restrictions in protein folding: an alpha-helix cannot be followed by a contiguous beta-strand

Using only hard-sphere repulsion, we investigated short polyalanyl chains for the presence of sterically imposed conformational constraints beyond the dipeptide level. We found that a central residue in a helical peptide cannot adopt dihedral angles from strand regions without encountering a steric collision. Consequently, an alpha-helical segment followed by a beta-strand segment must be connected by an intervening linker. This restriction was validated both by simulations and by seeking violations within proteins of known structure. In fact, no violations were found within an extensive database of high-resolution X-ray structures. Nature's exclusion of alpha-beta hybrid segments, fashioned from an alpha-helix adjoined to a beta-strand, is built into proteins at the covalent level. This straightforward conformational constraint has far-reaching consequences in organizing unfolded proteins and limiting the number of possible protein domains.