A comparison of position-specific score matrices based on sequence and structure alignments

Sequence comparison methods based on position-specific score matrices (PSSMs) have proven a useful tool for recognition of the divergent members of a protein family and for annotation of functional sites. Here we investigate one of the factors that affects overall performance of PSSMs in a PSI-BLAST search, the algorithm used to construct the seed alignment upon which the PSSM is based. We compare PSSMs based on alignments constructed by global sequence similarity (ClustalW and ClustalW-pairwise), local sequence similarity (BLAST), and local structure similarity (VAST). To assess performance with respect to identification of conserved functional or structural sites, we examine the accuracy of the three-dimensional molecular models predicted by PSSM-sequence alignments. Using the known structures of those sequences as the standard of truth, we find that model accuracy varies with the algorithm used for seed alignment construction in the pattern local-structure (VAST) > local-sequence (BLAST) > global-sequence (ClustalW). Using structural similarity of query and database proteins as the standard of truth, we find that PSSM recognition sensitivity depends primarily on the diversity of the sequences included in the alignment, with an optimum around 30-50% average pairwise identity. We discuss these observations, and suggest a strategy for constructing seed alignments that optimize PSSM-sequence alignment accuracy and recognition sensitivity.     

Predation of <Emphasis Type="Italic">Halyomorpha halys</Emphasis> (Hemiptera: Pentatomidae) from Web-Building Spiders Associated with Anthropogenic Dwellings

The brown marmorated stink bug, or Halyomorpha halys, is an invasive pest in North America and Europe that causes severe agricultural damage and nuisance problems for homeowners; and it is originally from China, Taiwan, and the Republic of Korea. While the natural enemy community of H. halys has been evaluated in several agroecosystems, it has not been examined where H. halys overwinters in anthropogenic structures. The aims of the current study were to evaluate 1) whether spider webs commonly found in the home and yard can successfully ensnare H. halys, 2) whether entanglement resulted in consumption by spiders inhabiting the webs, and 3) how frequently H. halys becomes entangled in webs under ambient conditions. To accomplish this, adult H. halys were introduced into webs in and near anthropogenic structures in West Virginia and Maryland, United States, and the behavior of spiders was observed for 5-min intervals at 0, 1, 2, and 24 h after introduction. In addition, a survey of webs was performed to determine the frequency with which spiders naturally capture H. halys inside buildings and in the landscape. Overall, the study found seven spider families in anthropogenic structures. Adult H. halys that were introduced into the webs of Theridiidae, Pholcidae, or Agelenidae had a greater than 50% chance of being ensnared and consumed. Adult H. halys were found naturally most often in webs of Theridiidae. Webs with a funnel or cob web architecture had the greatest probability of ensnaring H. halys, while those with orb structures resulted in the fewest caught. In the wild, 13–20% of spider webs contained dead H. halys. Our results suggest that spiders may be an important contributing factor for mortality of H. halys at overwintering sites, and spiders in or outside homes may help reduce nuisance problems caused by H. halys.     

Rapid Evaluation of Least-Squares and Minimum-Evolution Criteria on Phylogenetic Trees

We present fast new algorithms for evaluating trees with respectto least squares and minimum evolution (ME), the most commonlyused criteria for inferring phylogenetic trees from distancedata. The new algorithms include an optimal O(N2) time algorithmfor calculating the edge (branch or internode) lengths on atree according to ordinary or unweighted least squares (OLS);an O(N3) time algorithm for edge lengths under weighted leastsquares (WLS) including the Fitch-Margoliash method; and anoptimal O(N4) time algorithm for generalized least-squares (GLS)edge lengths (where N is the number of taxa in the tree). TheME criterion is based on the sum of edge lengths. Consequently,the edge lengths algorithms presented here lead directly toO(N2), O(N3), and O(N4) time algorithms for ME under OLS, WLS,and GLS, respectively. All of these algorithms are as fast asor faster than any of those previously published, and the algorithmsfor OLS and GLS are the fastest possible (with respect to orderof computational complexity). A major advantage of our new methodsis that they are as well adapted to multifurcating trees asthey are to binary trees. An optimal algorithm for determiningpath lengths from a tree with given edge lengths is also developed.This leads to an optimal O(N2) algorithm for OLS sums of squaresevaluation and corresponding O(N3) and O(N4) time algorithmsfor WLS and GLS sums of squares, respectively. The GLS algorithmis time-optimal if the covariance matrix is already inverted.The speed of each algorithm is assessed analytically—thespeed increases we calculate are confirmed by the dramatic speedincreases resulting from their implementation in PAUP* 4.0.The new algorithms enable far more extensive tree searches andstatistical evaluations (e.g., bootstrap, parametric bootstrap,or jackknife) in the same amount of time. Hopefully, the fastalgorithms for WLS and GLS will encourage the use of these criteriafor evaluating trees and their edge lengths (e.g., for approximatedivergence time estimates), since they should be more statisticallyefficient than OLS.     

Phycobilisome structure in the cyanobacteria Mastigocladus laminosus and Anabaena sp. PCC 7120.

Phycobilisomes of the cyanobacteria Mastigocladus laminosus and Anabaena sp. PCC7120 differ from typical tricylindrical, hemidiscoidal phycobilisomes in three respects. Firstly, size comparisons of the core-membrane linker phycobiliproteins (LCM) in different cyanobacteria by SDS/PAGE reveal an apparent molecular mass of 120 kDa for the LCM of M. laminosus and Anabaena sp. PCC7120. This observation suggests that the polypeptides of these species have four linker-repeat domains. Secondly, phycobilisomes of M. laminosus are shown to contain at least three, but most probably four, different rod-core linker polypeptides (LRC). These LRC, which attach the peripheral rods to the core and thereby make phycocyanin/allophycocyanin contacts, have been identified and characterized by N-terminal amino acid sequence analysis. Additionally, electron microscopy of phycobilisomes isolated from M. laminosus and Anabaena sp. PCC7120 reveals similar structures which differ from those of Calothrix sp. PCC7601 with their typical six, peripheral rods. Based upon protein-analytical results and a reinterpretation of the data of [Isono, T. & Katoh, T. (1987) Arch. Biochem. Biophys. 256, 317-324], we discuss structural implications of recent findings on the established hemidiscoidal model for the phycobilisomes of M. laminosus and Anabaena sp. PCC7120. Up to eight peripheral rods are suggested to radiate from a modified core substructure which contains two additional peripheral allophycocyanin hexamer equivalents that serve as the core-proximal discs for two peripheral rods.     

Arabidopsis Sec1/Munc18 Protein SEC11 Is a Competitive and Dynamic Modulator of SNARE Binding and SYP121-Dependent Vesicle Traffic

The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual “handshaking” mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.     

The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3.

trkB is a tyrosine protein kinase gene highly related to trk, a proto-oncogene that encodes a receptor for nerve growth factor (NGF) and neurotrophin-3 (NT-3). trkB expression is confined to structures of the central and peripheral nervous systems, suggesting it also encodes a receptor for neurotrophic factors. Here we show that brain-derived neurotrophic factor (BDNF) and NT-3, but not NGF, can induce rapid phosphorylation on tyrosine of gp145trkB, one of the receptors encoded by trkB. BDNF and NT-3 can induce DNA synthesis in quiescent NIH 3T3 cells that express gp145trkB. Cotransfection of plasmids encoding gp145trkB and BDNF or NT-3 leads to transformation of recipient NIH 3T3 cells. In these assays, BDNF elicits a response at least two orders of magnitude higher than NT-3. Finally, 125I-NT-3 binds to NIH 3T3 cells expressing gp145trkB; binding can be competed by NT-3 and BDNF but not by NGF. These findings indicate that gp145trkB may function as a neurotrophic receptor for BDNF and NT-3.     

Analysis of N-linked oligosaccharides: progress towards the characterisation of glycoprotein-linked carbohydrates

The covalent attachment of carbohydrate to proteins is a very common co- or post-translational event in the biosynthesis of glycoproteins. The type and heterogeneity of these oligosaccharides can affect a range of physico-chemical and biological properties of a glycoprotein. Thus the development of sensitive, reliable and robust analytical methods for carbohydrate analysis is important in the pharmaceutical industry, especially in the recombinant production of experimental and therapeutic glycoproteins. In this report we have reviewed methodology for the in-gel enzymatic release of N-linked oligosaccharides from glycoproteins separated by electrophoresis. These oligosaccharides are derivatised by reductive amination using 3-acetamido-6-aminoacridine (AA-Ac), a novel, highly fluorescent probe. A major advantage of this technique is that glycan derivatives are amenable to analysis by an array of chromatographic and mass spectrometric methods, allowing the resolution and characterisation of a wide variety of glycan structures. It is hoped that in due course the methodology described will be applied to proteomics studies, especially in identifying the role of carbohydrate in protein function and disease.     

Pretreatment with heat does not affect double-strand breaks DNA rejoining in Chlamydomonas reinhardtii

We aimed to clarify if heat pretreatment could protect Chlamydomonas reinhardtii cells from gamma rays DNA damaging action. It was studied whether: (1) heat pretreatment could accelerate DNA DSB rejoining; (2) chloroplast chaperones (HSP70B, HSP90C) could be involved in protection from radiation-induced DNA DSB.It was obtained that heat pretreatment (37–42 °C) induced minor DNA DSB levels which might be insufficient as signals for DNA DSB repair induction. No correlation between chaperones overproduction and DNA DSB rejoining was shown. These are probably the first data that HSP70B and HSP90C do not protect DNA against radiation-induced damage in a plant model system.     

Stereospecific induction of starfish oocyte maturation by (8R)-hydroxyeicosatetraenoic acid

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine. This induction of meiotic divisions can be triggered also by four fatty acids: 5,8,11-20:3; 5,8,11,14-20:4 (arachidonic acid); 6,9,12,15-20:4; 5,8,11,14,17-20:5, all other fatty acids being completely inactive. This maturation triggered by eicosanoids occurs in the micromolar range and is facilitated by the presence of calcium. A variety of arachidonic acid derivatives (esters, epoxides, etc.) and metabolites (cyclooxygenase and lipoxygenase products) has been tested; the biological activity is restricted to 8-hydroxyeicosatetraenoic acid (8-HETE), other mono- and poly-HETEs being completely inactive. Maturation triggered by 8-HETE occurs around 10 nM and is insensitive to the presence of calcium. 8-HETE methyl ester and 8-hydroperoxyeicosatetraenoic acid are able to induce maturation at higher concentrations. Both (8S) and (8R) stereoisomers have been tested; the biological activity is strictly restricted to the (8R) isomer. 8-HETE triggers a complete maturation, i.e. maturation-promoting factor appearance, germinal vesicle breakdown, emission of the polar bodies, and formation of a female pronucleus. (8R)-HETE, but not (8S)-HETE, triggers the typical decrease in cyclic AMP concentration induced by 1-methyladenine and the burst of protein phosphorylation associated with maturation. Starfish oocytes oxidize exogenous arachidonic acid into 8-HETE and other HETEs. 8-HETE was identified, after high pressure liquid chromatography purification, by gas chromatography mass spectrometry. Furthermore, it was found that the starfish oocytes only produce the (8R)-HETE isomer. This highly stereospecific induction of oocyte maturation by (8R)-HETE suggests that this fatty acid, or a very closely related fatty acid, may play a role in the transduction of the 1-methyladenine message at the plasma membrane level.     

Identification of a divalent metal cation binding site in herpes simplex virus 1 (HSV-1) ICP8 required for HSV replication

Herpes simplex virus 1 (HSV-1) ICP8 is a single-stranded DNA-binding protein that is necessary for viral DNA replication and exhibits recombinase activity in vitro. Alignment of the HSV-1 ICP8 amino acid sequence with ICP8 homologs from other herpesviruses revealed conserved aspartic acid (D) and glutamic acid (E) residues. Amino acid residue D1087 was conserved in every ICP8 homolog analyzed, indicating that it is likely critical for ICP8 function. We took a genetic approach to investigate the functions of the conserved ICP8 D and E residues in HSV-1 replication. The E1086A D1087A mutant form of ICP8 failed to support the replication of an ICP8 mutant virus in a complementation assay. E1086A D1087A mutant ICP8 bound DNA, albeit with reduced affinity, demonstrating that the protein is not globally misfolded. This mutant form of ICP8 was also recognized by a conformation-specific antibody, further indicating that its overall structure was intact. A recombinant virus expressing E1086A D1087A mutant ICP8 was defective in viral replication, viral DNA synthesis, and late gene expression in Vero cells. A class of enzymes called DDE recombinases utilize conserved D and E residues to coordinate divalent metal cations in their active sites. We investigated whether the conserved D and E residues in ICP8 were also required for binding metal cations and found that the E1086A D1087A mutant form of ICP8 exhibited altered divalent metal binding in an in vitro iron-induced cleavage assay. These results identify a novel divalent metal cation-binding site in ICP8 that is required for ICP8 functions during viral replication.