Identification of the gene (lgtG) encoding the lipooligosaccharide β chain synthesizing glucosyl transferase from Neisseria gonorrhoeae

The lipooligosaccharide from Neisseria gonorrhoeae (GC), consists of lipid A, an oligosaccharide core and three branches, α, β, and γ. We report the cloning of the gene (lgtG, lipooligosaccharide glycosyl transferase G) encoding the glucosyl transferase of GC that initiates the β chain which consists of a lactosyl moiety. This gene contains a homopolymeric tract of cytidine [poly(C)] and we demonstrate that changes in the number of Cs in poly(C) account for the variation of β chain expression in different GC strains. Biochemical analyses and mass spectrometry clearly attribute the reactivity of mAb 2C7 to the presence of the lactosyl β chain. In addition, we demonstrate that in the absence of the lactosyl group, a phosphoethanolamine is added to generate a new antigenic epitope as evidenced by the gain of reactivity to mAb 2-L1–8. These results show that, like the α chain, the β chain of lipooligosaccharide is subject to antigenic variation.

A 1.2-Å snapshot of the final step of bacterial cell wall biosynthesis

The cell wall imparts structural strength and shape to bacteria. It is made up of polymeric glycan chains with peptide branches that are cross-linked to form the cell wall. The cross-linking reaction, catalyzed by transpeptidases, is the last step in cell wall biosynthesis. These enzymes are members of the family of penicillin-binding proteins, the targets of β-lactam antibiotics. We report herein the structure of a penicillin-binding protein complexed with a cephalosporin designed to probe the mechanism of the cross-linking reaction catalyzed by transpeptidases. The 1.2-Å resolution x-ray structure of this cephalosporin bound to the active site of the bifunctional serine type d-alanyl-d-alanine carboxypeptidase/transpeptidase (EC 3.4.16.4) from Streptomyces sp. strain R61 reveals how the two peptide strands from the polymeric substrates are sequestered in the active site of a transpeptidase. The structure of this complex provides a snapshot of the enzyme and the bound cell wall components poised for the final and critical cross-linking step of cell wall biosynthesis.

Curvature Induced by Amyloplast Magnetophoresis in Protonemata of the Moss Ceratodon purpureus1

After gravistimulation of Ceratodon purpureus (Hedw.) Brid. protonemata in the dark, amyloplast sedimentation was followed by upward curvature in the wild-type (WT) and downward curvature in the wwr mutant (wrong way response). We used ponderomotive forces induced by high-gradient magnetic fields (HGMF) to simulate the effect of gravity and displace the presumptive statoliths. The field was applied by placing protonemata either between two permanent magnets at the edge of the gap, close to the edge of a magnetized ferromagnetic wedge, or close to a small (<1 mm) permanent magnet. Continuous application of an HGMF in all three configurations resulted in plastid displacement and induced curvature in tip cells of WT and wwr protonemata. WT cells curved toward the HGMF, and wwr cells curved away from the HGMF, comparable to gravitropism. Plastids isolated from protonemal cultures had densities ranging from 1.24 to 1.38 g cm−3. Plastid density was similar for both genotypes, but the mutant contained larger plastids than the WT. The size difference might explain the stronger response of the wwr protonemata to the HGMF. Our data support the plastid-based theory of gravitropic sensing and suggest that HGMF-induced ponderomotive forces can substitute for gravity.

Long term trends in the evolution of H(3) HA1 human influenza type A

We have studied the HA1 domain of 254 human influenza A(H3N2) virus genes for clues that might help identify characteristics of hemagglutinins (HAs) of circulating strains that are predictive of that strain’s epidemic potential. Our preliminary findings include the following. (i) The most parsimonious tree found requires 1,260 substitutions of which 712 are silent and 548 are replacement substitutions. (ii) The HA1 portion of the HA gene is evolving at a rate of 5.7 nucleotide substitutions/year or 5.7 × 10−3 substitutions/site per year. (iii) The replacement substitutions are distributed randomly across the three positions of the codon when allowance is made for the number of ways each codon can change the encoded amino acid. (iv) The replacement substitutions are not distributed randomly over the branches of the tree, there being 2.2 times more changes per tip branch than for non-tip branches. This result is independent of how the virus was amplified (egg grown or kidney cell grown) prior to sequencing or if sequencing was carried out directly on the original clinical specimen by PCR. (v) These excess changes on the tip branches are probably the result of a bias in the choice of strains to sequence and the detection of deleterious mutations that had not yet been removed by negative selection. (vi) There are six hypervariable codons accumulating replacement substitutions at an average rate that is 7.2 times that of the other varied codons. (vii) The number of variable codons in the trunk branches (the winners of the competitive race against the immune system) is 47 ± 5, significantly fewer than in the twigs (90 ± 7), which in turn is significantly fewer variable codons than in tip branches (175 ± 8). (viii) A minimum of one of every 12 branches has nodes at opposite ends representing viruses that reside on different continents. This is, however, no more than would be expected if one were to randomly reassign the continent of origin of the isolates. (ix) Of 99 codons with at least four mutations, 31 have ratios of non-silent to silent changes with probabilities less than 0.05 of occurring by chance, and 14 of those have probabilities <0.005. These observations strongly support positive Darwinian selection. We suggest that the small number of variable positions along the successful trunk lineage, together with knowledge of the codons that have shown positive selection, may provide clues that permit an improved prediction of which strains will cause epidemics and therefore should be used for vaccine production.

The representation of the ipsilateral visual field in human cerebral cortex

Previous studies of cortical retinotopy focused on influences from the contralateral visual field, because ascending inputs to cortex are known to be crossed. Here, functional magnetic resonance imaging was used to demonstrate and analyze an ipsilateral representation in human visual cortex. Moving stimuli, in a range of ipsilateral visual field locations, revealed activity: (i) along the vertical meridian in retinotopic (presumably lower-tier) areas; and (ii) in two large branches anterior to that, in presumptive higher-tier areas. One branch shares the anterior vertical meridian representation in human V3A, extending superiorly toward parietal cortex. The second branch runs antero-posteriorly along lateral visual cortex, overlying motion-selective area MT. Ipsilateral stimuli sparing the region around the vertical meridian representation also produced signal reductions (perhaps reflecting neural inhibition) in areas showing contralaterally driven retinotopy. Systematic sampling across a range of ipsilateral visual field extents revealed significant increases in ipsilateral activation in V3A and V4v, compared with immediately posterior areas V3 and VP. Finally, comparisons between ipsilateral stimuli of different types but equal retinotopic extent showed clear stimulus specificity, consistent with earlier suggestions of a functional segregation of motion vs. form processing in parietal vs. temporal cortex, respectively.

Pea formaldehyde-active class III alcohol dehydrogenase: common derivation of the plant and animal forms but not of the corresponding ethanol-active forms (classes I and P).

A plant class III alcohol dehydrogenase (or glutathione-dependent formaldehyde dehydrogenase) has been characterized. The enzyme is a typical class III member with enzymatic parameters and substrate specificity closely related to those of already established animal forms. Km values with the pea enzyme are 6.5 microM for NAD+, 2 microM for S-hydroxymethylglutathione, and 840 microM for octanol versus 9, 4, and 1200 microM, respectively, with the human enzyme. Structurally, the pea/human class III enzymes are closely related, exhibiting a residue identity of 69% and with only 3 of 23 residues differing among those often considered in substrate and coenzyme binding. In contrast, the corresponding ethanol-active enzymes, the long-known human liver and pea alcohol dehydrogenases, differ more (47% residue identities) and are also in functionally important active site segments, with 12 of the 23 positions exchanged, including no less than 7 at the usually much conserved coenzyme-binding segment. These differences affect functionally important residues that are often class-distinguishing, such as those at positions 48, 51, and 115, where the plant ethanol-active forms resemble class III (Thr, Tyr, and Arg, respectively) rather than the animal ethanol-active class I forms (typically Ser, His, and Asp, respectively). Calculations of phylogenetic trees support the conclusions from functional residues in subgrouping plant ethanol-active dehydrogenases and the animal ethanol-active enzymes (class I) as separate descendants from the class III line. It appears that the classical plant alcohol dehydrogenases (now called class P) have a duplicatory origin separate from that of the animal class I enzymes and therefore a paralogous relationship with functional convergence of their alcohol substrate specificity. Combined, the results establish the conserved nature of class III also in plants, and contribute to the molecular and functional understanding of alcohol dehydrogenases by defining two branches of plant enzymes into the system.

Glucocorticoid and progestin receptors are differently involved in the cooperation with a structural element of the mouse mammary tumor virus promoter.

We have previously characterized a regulatory element located between -294 and -200 within the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). This element termed AA element cooperates with the glucocorticoid response elements (GREs) for glucocorticoid activation. Here we show that in a MMTV LTR wild type context, the deletion of this element significantly reduces both glucocorticoid and progestin activation of the promoter. Deletion of the two most distal GREs forces the glucocorticoid receptor (GR) and the progestin receptor (PR) to bind the same response elements and results in a dramatic decrease in the inducibility of the MMTV promoter by the two hormones. The simultaneous deletion of the two distal GREs and of the AA element abolishes completely the glucocorticoid-induced activation of the promoter. In contrast it restores a significant level of progestin-induced activation. This different effect of the double deletion on glucocorticoid- and progestin-induced MMTV promoter activation is not cell specific because it is also observed, and is even stronger, when either GR or PR is expressed in the same cell line (NIH 3T3). This is the first description of a mutated MMTV promoter that, although retaining GREs, is activated by progestins and not by glucocorticoids. This suggests a different functional cooperation between protein(s) interacting with the AA element and GR or PR. Cotransfections with constructs containing wild-type or mutated MMTV LTR with either PR lacking its C-terminal domain or GR/PR chimeras in which the N-terminal domains have been exchanged demonstrate that the N-terminal domains of the receptors specify the different behavior of GR and PR regarding the AA element.

The genetic link between the Chinese bamboo partridge (Bambusicola thoracica) and the chicken and junglefowls of the genus Gallus.

Further comparison of mitochondrial control-region DNA base sequences of 16 avian species belonging to the subfamily Phasianinae revealed the following: (i) Generalized perdicine birds (quails and partridges) are of ancient lineages. Even the closest pair, the common quail (Coturnix coturnix japonica) and the Chinese bamboo partridge (Bambusicola thoracica), maintained only 85.71% identity. (ii) The 12 species of phasianine birds previously and presently studied belonged to three distinct branches. The first branch was made exclusively of members of the genus Gallus, while the second branch was made of pheasants of the genera Phasianus, Chrysolophus, and Syrmaticus. Gallopheasants of the genus Lophura were distant cousins to these pheasants. The great argus (Argusianus argus) and peafowls of the genus Pavo constituted the third branch. The position of peacock-pheasants of the genus Polyplectron in the third branch was similar to that of the genus Lophura in the second branch. Members of the fourth phasianine branch, such as tragopans and monals, were not included in the present study. (iii) The one perdicine species, Bambusicola thoracica, was more closely related to phasianine genera Gallus and Pavo than to members of other perdicine genera. The above might indicate that Bambusicola belong to one-stem perdicine lineage that later splits into two sublineages that yielded phasianine birds, one evolving to Gallus, and the other differentiating toward Pavo and its allies.

On the magnitude of the electrostatic contribution to ligand-DNA interactions.

A model based on the nonlinear Poisson-Boltzmann equation is used to study the electrostatic contribution to the binding free energy of a simple intercalating ligand, 3,8-diamino-6-phenylphenanthridine, to DNA. We find that the nonlinear Poisson-Boltzmann model accurately describes both the absolute magnitude of the pKa shift of 3,8-diamino-6-phenylphenanthridine observed upon intercalation and its variation with bulk salt concentration. Since the pKa shift is directly related to the total electrostatic binding free energy of the charged and neutral forms of the ligand, the accuracy of the calculations implies that the electrostatic contributions to binding are accurately predicted as well. Based on our results, we have developed a general physical description of the electrostatic contribution to ligand-DNA binding in which the electrostatic binding free energy is described as a balance between the coulombic attraction of a ligand to DNA and the disruption of solvent upon binding. Long-range coulombic forces associated with highly charged nucleic acids provide a strong driving force for the interaction of cationic ligands with DNA. These favorable electrostatic interactions are, however, largely compensated for by unfavorable changes in the solvation of both the ligand and the DNA upon binding. The formation of a ligand-DNA complex removes both charged and polar groups at the binding interface from pure solvent while it displaces salt from around the nucleic acid. As a result, the total electrostatic binding free energy is quite small. Consequently, nonpolar interactions, such as tight packing and hydrophobic forces, must play a significant role in ligand-DNA stability.

Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications.

Universal trees based on sequences of single gene homologs cannot be rooted. Iwabe et al. [Iwabe, N., Kuma, K.-I., Hasegawa, M., Osawa, S. & Miyata, T. (1989) Proc. Natl. Acad. Sci. USA 86, 9355-9359] circumvented this problem by using ancient gene duplications that predated the last common ancestor of all living things. Their separate, reciprocally rooted gene trees for elongation factors and ATPase subunits showed Bacteria (eubacteria) as branching first from the universal tree with Archaea (archaebacteria) and Eucarya (eukaryotes) as sister groups. Given its topical importance to evolutionary biology and concerns about the appropriateness of the ATPase data set, an evaluation of the universal tree root using other ancient gene duplications is essential. In this study, we derive a rooting for the universal tree using aminoacyl-tRNA synthetase genes, an extensive multigene family whose divergence likely preceded that of prokaryotes and eukaryotes. An approximately 1600-bp conserved region was sequenced from the isoleucyl-tRNA synthetases of several species representing deep evolutionary branches of eukaryotes (Nosema locustae), Bacteria (Aquifex pyrophilus and Thermotoga maritima) and Archaea (Pyrococcus furiosus and Sulfolobus acidocaldarius). In addition, a new valyl-tRNA synthetase was characterized from the protist Trichomonas vaginalis. Different phylogenetic methods were used to generate trees of isoleucyl-tRNA synthetases rooted by valyl- and leucyl-tRNA synthetases. All isoleucyl-tRNA synthetase trees showed Archaea and Eucarya as sister groups, providing strong confirmation for the universal tree rooting reported by Iwabe et al. As well, there was strong support for the monophyly (sensu Hennig) of Archaea. The valyl-tRNA synthetase gene from Tr. vaginalis clustered with other eukaryotic ValRS genes, which may have been transferred from the mitochondrial genome to the nuclear genome, suggesting that this amitochondrial trichomonad once harbored an endosymbiotic bacterium.

The Significance of the Disputed Territory in Bechuanaland 1887-1893

British Imperial policy in Southern Africa in the last three decades of the nineteenth century oscillated between two extremes. It began in the early 1870's with Lord Kimberley's attempt to effect confederation as a means of devolving Imperial responsibility and expenditure. It ended in 1899 with Britain's active intervention against the Boers. For most of the remaining years of those decades a middle course was adopted while the British Government struggled to reconcile its diverse political interests. Strategy, supremacy, economy, humanitarianism, and recognition of colonial aspirations were all at one time or another, in varying degrees, motivating forces behind Imperial policy. Many historians have pointed out how incompatible many of these ends were and how the attempt to pursue them all at once almost inevitably ended in at least one of them being sacrificed on the way. This study focusses on a relatively minor problem over a period of about seven years. It attempts to show how the British Government tried to reconcile, in this case, the predominant motives of economy and supremacy. The problem of the Disputed Territory now seems like a small fish in a big ocean because non the great hopes and fears that it raised were ever realized. But the anticlimactic nature of the outcome of events should not be allowed to conceal two important points: first, that the problem loomed large at the time in the eyes of the Imperial Government; and second, that in the case of its policy towards the Disputed Territory, the Government gained a greater degree of success in trying to reconcile seemingly incompatible ends than it did in many other instances.

The adoption of IPSASs in the Iberian Peninsula: the perspective of the standar-setters

The last decades have been fruitful in reforms in public sector accounting across the world, namely moving from cash-based into accrual-based regimes. In this process of bringing public sector accounting close to business accounting, International Public Sector Accounting Standards (IPSASs) have been developed and adopted in several countries. In the EU context, public sector harmonization among member-States is currently being considered, namely via the development of European Public Sector Accounting Standards (EPSASs). Both IPSASs and EPSASs are understood as, among other things, important to contribute for a more informative and transparent financial reporting of public sector entities and governments. Moreover they are expected to approximate public sector accounting and the National Accounts, hence allowing for more reliable information to monitor fiscal discipline among EU countries. The Iberian countries, after using accrual accounting in the public sector for more than twenty years (Spain from middle 1980s and Portugal from 1990s), have acknowledged the need to embark in an international harmonization process adopting IPSASs, particularly after the adoption of IFRSs in the business sector, which was creating some difficulties for consolidated accounts. Spain has passed the Chart of Accounts for the Public Sector through the Order EHA/1037/2010, which is adapted to IPSASs; Portugal has just passed Law-decree 192/2015, September 11, and is expecting to start implementation in 2017. In both countries a central role in this reform has been assumed by the national standard-setters for public sector accounting. Based on the “Actor Network Theory” to help understanding how and why organizations interact and how this interaction could have an impact on their choice of accounting policies, this paper aims to analyse, from the Iberian public sector accounting standard-setters perspective, how the adoption/adaptation of IPSASs has been considered and developed in these countries. Research questions to be considered are the following, which will be asked in interviews to the members of the standard-setting committees in both Portugal and Spain: • What were the driving forces leading to the decision to adopt and implement IPSASs? • Which other governmental bodies’ experiences, if any, were considered in the adoption of IPSASs? • What specific steps were/are being undertaken to prepare for the conversion from the existing system to IPSASs? • What were/are the institutional challenges faced/expected to face? • What assistance have been received from IFAC and/or IPSASB, or from other sources, if any, throughout the adoption/implementation? • How can the success of the adoption/implementation be characterised? • What benefits can/have the government derive/derived from the adoption and implementation of IPSASs? The paper is expected to contribute to the understanding of the issues underlying the process of embarking in public sector accounting reforms towards IPSASs.

Building up European leadership - an assessment of the recent process. CHALLENGE EUROPE Issue 22 - Challenges and new beginnings: Priorities for the EU's new leadership, September 2014.

Europe is in need of a new leadership capacity able to recreate stronger European unity in the external and internal fronts. Otherwise, anti-European forces will increase their influence and presence in European governments and EU institutions with large implications for the direction of European integration. This will be the central concern in making a first short assessment of the recent process of building European leadership capacity for the next five years to come. This assessment will particularly focus on the choice of the President of the European Commission, of the President of the European Council and, finally of the members of the European Commission.

"Creeping" civil war in the North Caucasus. OSW Commentary No. 50, 2011-03-15

Over the last year, the situation in Russia’s North Caucasus has become further destabilised. Attacks and armed clashes happen daily, and destabilisation is spreading to an increasingly large area. The extent of violence in the region is so great that it can already be stated that a de facto civil war is taking place, the warring parties being the Islamic armed underground movement which operates under the banner of the so-called Emirate of the North Caucasus, and the secular governments of the individual republics, who are supported by local and federal branches of the Russian Federation’s Interior Ministry and Federal Security Service. Moscow has no idea how to successfully tackle the Caucasus rebellion. Force has proved to be costly and unproductive, while the attempts made since early 2010 to integrate the region with the rest of Russia by implementing development programmes have not brought the desired results, because of widespread corruption and faint interest from businessmen who are afraid to invest in such an unsafe region. A growing problem for Moscow, particularly for the prestige of the state, is attacks by militants on areas near Sochi, where the 2014 Winter Olympics is to take place. It must be assumed that over the next 3 years before the Olympics, Moscow’s priority in the region will be to ensure the safety of Olympic preparations, and then the games themselves. It cannot be ruled out that the North Caucasus Federal District with its ‘troubled republics’ will be surrounded by a kind of cordon sanitaire (Sochi is situated in the neighbouring Southern Federal District). This could in turn strengthen these republics’ isolation, maintain the state of permanent instability, and postpone the prospects of solving the region’s acute economic and social problems.

The 'tribal areas' of the Caucasus: The North Caucasus - an enclave of 'alien civilisation' within the Russian Federation. OSW Study 34/2010

Militant Islam is currently the greatest threat to security and stability in the Russian part of the Caucasus. However, even though the armed Islamic underground is capable of organising terrorist attacks and carrying out actions of sabotage, it seems too weak to bring about any change in the Caucasus’s political status quo. Besides, militant Islam is merely a symptom (albeit the most radical and spectacular) of a much wider process, namely the widening civilisational gap between Russia and the North Caucasus, initiated by the collapse of the Soviet Union. The key elements of this process are as follows: the spontaneous re-Islamisation of social life and the dynamic growth of Islam's political influence; the de-Russification of the region; and the ongoing marginalisation of secular intellectuals. As a result, the North Caucasus, and principally Chechnya, Ingushetia and Dagestan, are turning into an enclave separated from the rest of the Russian Federation by a growing civilisational gap, and becoming increasingly different from the rest of Russia. This situation may recall the tribal areas of Pakistan inhabited by Pashtuns (FATA) along the Afghan border.