Molecular phylogeny of the fungus gnat tribe Exechiini (Mycetophilidae, Diptera)

The phylogenetic relationships within the fungus gnat tribe Exechiini have been left unattended for many years. Recent studies have not shed much light on the intergeneric relationship within the tribe. Here the first attempt to resolve the phylogeny of the tribe Exechiini using molecular markers is presented. The nuclear 18S and the mitochondrial 16S, and cytochrome oxidase subunit I (COI) genes were successfully sequenced for 20 species representing 15 Exechiini genera and five outgroup genera. Bayesian, maximum parsimony and maximum likelihood analyses revealed basically congruent tree topologies and the monophyly of Exechiini, including the genus Cordyla , is confirmed. The molecular data corroborate previous morphological studies in several aspects. Cordyla is found in a basal clade together with Brachypeza , Pseudorymosia and Stigmatomeria . The splitting of the genera Allodiopsis s.l. and Brevicornu s.l. as well as the sistergroup relationship of Exechia and Exechiopsis is also supported. The limited phylogenetic information provided by morphological characters is mirrored in the limited resolution of the molecular markers used in this study. Short internal and long-terminal branches obtained may indicate a rapid radiation of the Exechiini genera during a short evolutionary period.     

Intraspecific evolution ofMicroseris pygmaea (Asteraceae,Lactuceae) analyzed by cosegregation of phenotypic characters (QTLs) and molecular markers (RAPDs)

The Chilean annual,Microseris pygmaea, has differentiated in distinct coastal and inland series of populations after long-distance dispersal from western North America. Two plants from the most diverse biotypes were crossed, a large F₂ was raised and analysed for segregation of 30 phenotypic characters. Segregation of molecular markers (47 RAPDs, 1 RFLP, 2 isozymes) was determined in a subpopulation of 45 plants which include all extremes for the phenotypic characters. 32 marker/character cosegregations were significant at the 1% level in t-tests between dominant and homozygous recessive marker genotypes. Considering linkage among markers and pleiotropy of certain marker loci, the number of independent quantitative trait loci (QTLs) is reduced to about 18. Interactions among 2 or 3 QTLs affecting one character have been characterized. The phenotypic differentiation ofM. pygmaea during its evolution from a single founder individual begins to be understood at the level of single-gene mutants.     

The A-kinase Anchoring Protein GSKIP Regulates GSK3β Activity and Controls Palatal Shelf Fusion in Mice

A-kinase anchoring proteins (AKAPs) represent a family of structurally diverse proteins, all of which bind PKA. A member of this family is glycogen synthase kinase 3β (GSK3β) interaction protein (GSKIP). GSKIP interacts with PKA and also directly interacts with GSK3β. The physiological function of the GSKIP protein in vivo is unknown. We developed and characterized a conditional knock-out mouse model and found that GSKIP deficiency caused lethality at birth. Embryos obtained through Caesarean section at embryonic day 18.5 were cyanotic, suffered from respiratory distress, and failed to initiate breathing properly. Additionally, all GSKIP-deficient embryos showed an incomplete closure of the palatal shelves accompanied by a delay in ossification along the fusion area of secondary palatal bones. On the molecular level, GSKIP deficiency resulted in decreased phosphorylation of GSK3β at Ser-9 starting early in development (embryonic day 10.5), leading to enhanced GSK3β activity. At embryonic day 18.5, GSK3β activity decreased to levels close to that of wild type. Our findings reveal a novel, crucial role for GSKIP in the coordination of GSK3β signaling in palatal shelf fusion.     

Spiralian phylogenomics supports the resurrection of Bryozoa comprising Ectoprocta and Entoprocta

Phylogenetic analyses based on 79 ribosomal proteins of 38 metazoans, partly derived from 6 new expressed sequence tag projects for Ectoprocta, Entoprocta, Sipuncula, Annelida, and Acanthocephala, indicate the monophyly of Bryozoa comprising Ectoprocta and Entoprocta, 2 taxa that have been separated for more than a century based on seemingly profound morphological differences. Our results also show that bryozoans are more closely related to Neotrochozoa, including molluscs and annelids, than to Syndermata, the latter comprising Rotifera and Acanthocephala. Furthermore, we find evidence for the position of Sipuncula within Annelida. These findings suggest that classical developmental and morphological key characters such as cleavage pattern, coelomic cavities, gut architecture, and body segmentation are subject to greater evolutionary plasticity than traditionally assumed.     

Expression of a bacterial sucrose phosphorylase in potato tubers results in a glucose-independent induction of glycolysis

Sugars are not only metabolic substrates: they also act as signals that regulate the metabolism of plants. Previously, we found that glycolysis is induced in transgenic tubers expressing a yeast invertase in the cytosol but not in those expressing invertase in the apoplast. This suggests that either the low level of sucrose, the increased formation of cytosolic glucose or the increased levels of metabolites downstream of the sucrose cleavage is responsible for the induction of glycolysis in storage organs. In order to discriminate between these possibilities, we cloned and expressed a bacterial sucrose phosphorylase gene from Pseudomonas saccharophila in potato tubers. Due to the phosphorolytic cleavage of sucrose, formation of glucose was circumvented, thus allowing assessment of the importance of cytosolic glucose – and, by implication, flux through hexokinase – in glycolytic induction. Expression of sucrose phosphorylase led to: (i) a decrease in sucrose content, but no decrease in glucose or fructose; (ii) a decrease in both starch accumulation and tuber yield; (iii) increased levels of glycolytic metabolites; (iv) an induction of the activities of key enzymes of glycolysis; and (v) increased respiratory activity. We conclude that the induction of glycolysis in heterotrophic tissues such as potato tubers occurs via a glucose‐independent mechanism.     

Evidence for anextra-cellular function for protein kinase A

In addition to itsintra-cellular functions, cAMP-dependent protein kinase (PKA) may well have anextra-cellular regulatory role in blood. This suggestion is based on the following experimental findings: (a) Physiological stimulation of blood platelets brings about a specific release of PKA, together with its co-substrates ATP and Mg⁺⁺; (b) In human serum, an endogenous phosphorylation of one protein (p75, Mr 75 kDa) occurs; this phosphorylation is enhanced by addition of cAMP and blocked by the Walsh-Krebs specific PKA inhibitor; (c) No endogenous phosphorylation of p75 occurs in human plasma devoid of platelets, but the selective labeling of p75 can be reproduced by adding to plasma the pure catalytic subunit of PKA; (d) p75 was shown to be vitronectin (V), a multifunctional protein implicated in processes associated with platelet activation, and thus a protein whose function may require modulation for control; (e) The phosphorylation of vitronectin occurs at one site (Ser³⁷⁸) which, at physiological pH, is buried in its two-chain form (V₆₅₊₁₀) but becomes exposed in the presence of glycosaminoglycans (GAGs) e.g. heparin or heparan sulfate. Such a transconformation may be used for targeting the PKA phosphorylation to vitronectin molecules bound to GAGs, for example in the extracellular matrix or on cell surfaces; (f) From the biochemical point of view (Km values and physiological concentrations) the phosphorylation of vitronectin can take place at the locus of a hemostatic event; (g) The phosphorylation of Ser³⁷⁸ in vitronectin alters its function, since it significantly reduces its ability to bind the inhibitor-1 of plasminogen activator(s) (PAI-1). Physiologically, this functional modulation may be involved in unleashing PAI-1, allowing its translocation to control the inhibitory function of PAI-1 and, through it, regulating the conversion of plasminogen to active plasmin.Dedicated to Edmond H. Fischer and Edwin G. Krebs, with gratitude for teaching us the right measure of thoroughness and vision in research.     

Guaianolides from two subspecies of Amphoricarpos neumayeri from Montenegro

Quantitative (1)H NMR measurements revealed delta(11(13)) sesquiterpene gamma-lactones as the main constituents ( >or= 1% per weight of dried plant material) in the crude extracts of the aerial parts of Amphoricarpos neumayeri ssp. neumayeri and ssp. murbeckii from mountains Orjen and Visitor (Montenegro), respectively. Preparative silica gel chromatography afforded thirteen guai-11(13)-en-12,6alpha-olides, named amphoricarpolides (1-13), with the same relative (1alphaH,4betaH,5alphaH,7betaH) configuration of the basic skeleton. The common structural feature of lactones 2-13 was 3beta,15-dioxygenation pattern. The only exception was 1 (3-deoxyamphoricarpolide), containing a single oxygen substituent (15-OH). Eight of them exhibited an additional oxygen substituent, 9beta-OH (5 and 6), 2alpha-OH (8-12), or 2alpha-OAc (13). Compound 7 was epoxydated at 10alpha(14)-position, whereas the remaining lactones contained a 10(14) double bond.     

Mutations of the PC2 substrate binding pocket alter enzyme specificity

By taking advantage of the recently published furin structure, whose catalytic domain shares high homology with other proprotein convertases, we designed mutations in the catalytic domain of PC2, altering residues Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. In order to investigate the determinants of PC2 specificity, we have tested the mutated enzymes against a set of proenkephalin-derived substrates, as well as substrates representing Arg, Ala, Leu, Phe, and Glu positional scanning variants of a peptide B-derived substrate. We found that the exchange of the Ser206 residue with Arg or Lys led to a total loss of activity. Increased positive charge of the substrate generally resulted in an increased specificity constant. Most intriguingly, the RE281GR mutation, corresponding to a residue placed distantly in the S6 pocket, evoked the largest changes in the specificity pattern. The D278E and N356S mutations resulted in distinct alterations in PC2 substrate preferences. However, when other residues that distinguish PC2 from other convertases were substituted with PC1-like or furin-like equivalents, there was no significant alteration of the PC2 specificity pattern, suggesting that the overall structure of the substrate binding cleft rather than individual residues specifies substrate binding.     

CEACAM1 recognition by bacterial pathogens is species-specific

Background  

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an immunoglobulin (Ig)-related glycoprotein, serves as cellular receptor for a variety of Gram-negative bacterial pathogens associated with the human mucosa. In particular, Neisseria gonorrhoeae, N. meningitidis, Moraxella catarrhalis, and Haemophilus influenzae possess well-characterized CEACAM1-binding adhesins. CEACAM1 is typically involved in cell-cell attachment, epithelial differentiation, neovascularisation and regulation of T-cell proliferation, and is one of the few CEACAM family members with homologues in different mammalian lineages. However, it is unknown whether bacterial adhesins of human pathogens can recognize CEACAM1 orthologues from other mammals.