Inactive conformation of the serpin α1-antichymotrypsin indicates two-stage insertion of the reactive loop: Implications for inhibitory function and conformational disease

The serpins are a family of proteinase inhibitors that play a central role in the control of proteolytic cascades. Their inhibitory mechanism depends on the intramolecular insertion of the reactive loop into β-sheet A after cleavage by the target proteinase. Point mutations within the protein can allow aberrant conformational transitions characterized by β-strand exchange between the reactive loop of one molecule and β-sheet A of another. These loop-sheet polymers result in diseases as varied as cirrhosis, emphysema, angio-oedema, and thrombosis, and we recently have shown that they underlie an early-onset dementia. We report here the biochemical characteristics and crystal structure of a naturally occurring variant (Leu-55–Pro) of the plasma serpin α1-antichymotrypsin trapped as an inactive intermediate. The structure demonstrates a serpin configuration with partial insertion of the reactive loop into β-sheet A. The lower part of the sheet is filled by the last turn of F-helix and the loop that links it to s3A. This conformation matches that of proposed intermediates on the pathway to complex and polymer formation in the serpins. In particular, this intermediate, along with the latent and polymerized conformations, explains the loss of activity of plasma α1-antichymotrypsin associated with chronic obstructive pulmonary disease in patients with the Leu-55–Pro mutation.

Identification of the proton pathway in bacterial reaction centers: Both protons associated with reduction of QB to QBH2 share a common entry point

The reaction center from Rhodobacter sphaeroides uses light energy for the reduction and protonation of a quinone molecule, QB. This process involves the transfer of two protons from the aqueous solution to the protein-bound QB molecule. The second proton, H+(2), is supplied to QB by Glu-L212, an internal residue protonated in response to formation of QA− and QB−. In this work, the pathway for H+(2) to Glu-L212 was studied by measuring the effects of divalent metal ion binding on the protonation of Glu-L212, which was assayed by two types of processes. One was proton uptake from solution after the one-electron reduction of QA (DQA→D+QA−) and QB (DQB→D+QB−), studied by using pH-sensitive dyes. The other was the electron transfer kAB(1) (QA−QB→QAQB−). At pH 8.5, binding of Zn2+, Cd2+, or Ni2+ reduced the rates of proton uptake upon QA− and QB− formation as well as kAB(1) by ≈an order of magnitude, resulting in similar final values, indicating that there is a common rate-limiting step. Because D+QA− is formed 105-fold faster than the induced proton uptake, the observed rate decrease must be caused by an inhibition of the proton transfer. The Glu-L212→Gln mutant reaction centers displayed greatly reduced amplitudes of proton uptake and exhibited no changes in rates of proton uptake or electron transfer upon Zn2+ binding. Therefore, metal binding specifically decreased the rate of proton transfer to Glu-L212, because the observed rates were decreased only when proton uptake by Glu-L212 was required. The entry point for the second proton H+(2) was thus identified to be the same as for the first proton H+(1), close to the metal binding region Asp-H124, His-H126, and His-H128.

Equilibrium distributions of microsatellite repeat length resulting from a balance between slippage events and point mutations

We describe and test a Markov chain model of microsatellite evolution that can explain the different distributions of microsatellite lengths across different organisms and repeat motifs. Two key features of this model are the dependence of mutation rates on microsatellite length and a mutation process that includes both strand slippage and point mutation events. We compute the stationary distribution of allele lengths under this model and use it to fit DNA data for di-, tri-, and tetranucleotide repeats in humans, mice, fruit flies, and yeast. The best fit results lead to slippage rate estimates that are highest in mice, followed by humans, then yeast, and then fruit flies. Within each organism, the estimates are highest in di-, then tri-, and then tetranucleotide repeats. Our estimates are consistent with experimentally determined mutation rates from other studies. The results suggest that the different length distributions among organisms and repeat motifs can be explained by a simple difference in slippage rates and that selective constraints on length need not be imposed.

Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity

Leishmania parasites lack a purine biosynthetic pathway and depend on surface nucleoside and nucleobase transporters to provide them with host purines. Leishmania donovani possess two closely related genes that encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the natural substrates. In this study, we have characterized a drug-resistant clonal mutant of L. donovani (TUBA5) that is deficient in LdNT1 transport and consequently resistant to tubercidin. In TUBA5 cells, the LdNT1.2 genes had the same sequence as wild-type cells. However, because LdNT1.2 mRNA is not detectable in either wild-type or TUBA5 promastigotes, LdNT1.2 does not contribute to nucleoside transport in this stage of the life cycle. In contrast, the TUBA5 cells were compound heterozygotes at the LdNT1.1 locus containing two mutant alleles that encompassed distinct point mutations, each of which impaired transport function. One of the mutant LdNT1.1 alleles encoded a G183D substitution in predicted TM 5, and the other allele contained a C337Y change in predicted TM 7. Whereas G183D and C337Y mutants had only slightly elevated adenosine Km values, the severe impairment in transport resulted from drastically (≈20-fold) reduced Vmax values. Because these transporters were correctly targeted to the plasma membrane, the reduction in Vmax apparently resulted from a defect in translocation. Strikingly, G183 was essential for pyrimidine nucleoside but not adenosine transport. A mutant transporter with a G183A substitution had an altered substrate specificity, exhibiting robust adenosine transport but undetectable uridine uptake. These results suggest that TM 5 is likely to form part of the nucleoside translocation pathway in LdNT1.1

Two mutant prion proteins expressed in cultured cells acquire biochemical properties reminiscent of the scrapie isoform.

Prion diseases are a group of fatal neurodegenerative disorders that are unique in being infectious, genetic, and sporadic in origin. Infectious cases are caused by prions, which are composed primarily of PrPSc, a posttranslationally modified isoform of the normal cellular prion protein PrPC. Inherited cases are linked to insertional or point mutations in the host gene encoding PrPC. To investigate the molecular mechanisms underlying inherited prion diseases, we have constructed stably transfected Chinese hamster ovary cells that express mouse PrPs homologous to two human PrPs associated with familial Creutzfeldt-Jakob disease. One mouse PrP molecule carries a Glu-->Lys substitution at codon 199, and the other carries an insertion of six additional octapeptide repeats between codons 51 and 90. We find that both of these mutant PrPs display several biochemical hallmarks of PrPSc when synthesized in cell culture. Unlike wild-type PrP, the mutant proteins are detergent insoluble and are relatively resistant to digestion by proteinase K, yielding an N-terminally truncated core fragment of 27-30 kDa. Pulse-chase labeling experiments demonstrate that these properties are acquired posttranslationally, and are accompanied by increased metabolic stability of the protein. Our results provide the first evidence that a molecule with properties reminiscent of PrPSc can be generated de novo in cultured cells.

Disruption of RB/E2F-1 interaction by single point mutations in E2F-1 enhances S-phase entry and apoptosis.

The retinoblastoma protein (RB) has been proposed to function as a negative regulator of cell proliferation by complexing with cellular proteins such as the transcription factor E2F. To study the biological consequences of the RB/E2F-1 interaction, point mutants of E2F-1 which fail to bind to RB were isolated by using the yeast two-hybrid system. Sequence analysis revealed that within the minimal 18-amino acid peptide of E2F-1 required for RB binding, five residues, Tyr (position 411), Glu (419), and Asp-Leu-Phe (423-425), are critical. These amino acids are conserved among the known E2F family members. While mutation of any of these five amino acids abolished binding to RB, all mutants retained their full transactivation potential. Expression of mutated E2F-1, when compared with that of wild-type, significantly accelerated entry into S phase and subsequent apoptosis. These results provide direct genetic evidence for the biological significance of the RB/E2F interaction and strongly suggest that the interplay between RB and E2F is critical for proper cell cycle progression.

A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense.

In this paper we report a recessive mutation, immune deficiency (imd), that impairs the inducibility of all genes encoding antibacterial peptides during the immune response of Drosophila. When challenged with bacteria, flies carrying this mutation show a lower survival rate than wild-type flies. We also report that, in contrast to the antibacterial peptides, the antifungal peptide drosomycin remains inducible in a homozygous imd mutant background. These results point to the existence of two different pathways leading to the expression of two types of target genes, encoding either the antibacterial peptides or the antifungal peptide drosomycin.

Stabilisation of low softening point petroleum pitch fibres by iodine treatment

A method using iodine has been developed for the stabilisation of low softening point (SP) pitch fibres that avoids air stabilisation in the production of carbon fibres (CF). The interaction between iodine and petroleum pitches has been studied by following the changes in the hydrogen content, aromatic or aliphatic, during the heat treatment of iodine-treated pitch fibres. Two low SP petroleum pitches were used and the iodine-treated pitch fibres were analysed by TGA, DSC, DRIFT, XPS and SEM. The results confirm that using this novel method pitches with low SP can be used to prepare CF with two advantages, compared with conventional methods. The stabilisation time is considerably reduced and treatments to increase the SP, usually required when low SP pitches are used to prepare CF, can be avoided.

Discontinuity spacing analysis in rock masses using 3D point clouds

The complete characterization of rock masses implies the acquisition of information of both, the materials which compose the rock mass and the discontinuities which divide the outcrop. Recent advances in the use of remote sensing techniques – such as Light Detection and Ranging (LiDAR) – allow the accurate and dense acquisition of 3D information that can be used for the characterization of discontinuities. This work presents a novel methodology which allows the calculation of the normal spacing of persistent and non-persistent discontinuity sets using 3D point cloud datasets considering the three dimensional relationships between clusters. This approach requires that the 3D dataset has been previously classified. This implies that discontinuity sets are previously extracted, every single point is labeled with its corresponding discontinuity set and every exposed planar surface is analytically calculated. Then, for each discontinuity set the method calculates the normal spacing between an exposed plane and its nearest one considering 3D space relationship. This link between planes is obtained calculating for every point its nearest point member of the same discontinuity set, which provides its nearest plane. This allows calculating the normal spacing for every plane. Finally, the normal spacing is calculated as the mean value of all the normal spacings for each discontinuity set. The methodology is validated through three cases of study using synthetic data and 3D laser scanning datasets. The first case illustrates the fundamentals and the performance of the proposed methodology. The second and the third cases of study correspond to two rock slopes for which datasets were acquired using a 3D laser scanner. The second case study has shown that results obtained from the traditional and the proposed approaches are reasonably similar. Nevertheless, a discrepancy between both approaches has been found when the exposed planes members of a discontinuity set were hard to identify and when the planes pairing was difficult to establish during the fieldwork campaign. The third case study also has evidenced that when the number of identified exposed planes is high, the calculated normal spacing using the proposed approach is minor than those using the traditional approach.

Eastern Point, East Gloucester, Massachusetts, 1888 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Lands of The Eastern Point Associates, East Gloucester, Mass., [by] William H. Foss, engr. and supt. It was published by Forbes Co., Mar. 1st, 1888. Scale [ca. 1:3,550]. Cadastral map showing roads, property lot numbers, dimensions, and areas. Covers Eastern Point and part of East Gloucester, Gloucester, Massachusetts. Relief is shown by hachures. It includes two vignettes. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Massachusetts State Plane Coordinate System, Mainland Zone (in Feet) (Fipszone 2001). All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, or other information associated with the principal map. This layer is part of a selection of digitally scanned and georeferenced historic maps of Massachusetts from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of regions, originators, ground condition dates (1755-1922), scales, and purposes. The digitized selection includes maps of: the state, Massachusetts counties, town surveys, coastal features, real property, parks, cemeteries, railroads, roads, public works projects, etc.

Assessing the Single Supervisory Mechanism: passing the point of no return for Europe's banking union. Egmont Paper No. 58, June 2013

From the Executive Summary. Europe’s financial and sovereign debt crises have become increasingly interconnected. In order to break the negative feedback loop between the two, the EU has decided to create a common supervisory framework for the banking sector: the Single Supervisory Mechanism (SSM). The SSM will involve a supervisory system including both the national supervisors and the European Central Bank (ECB). By endowing the ECB with supervisory authority over a major part of the European banking sector, the SSM’s creation will result in a shake-up of the way in which the European financial sector is being supervised. Under the right circumstances, this could be a major step forward in addressing Europe’s interconnected crises.

Historic turning point or just another chapter? The results of a foreign-policy summit. EPC Post-Summit Analysis, 24 March 2014

The March 2014 European Council could enter the history books as a turning point, not only in the EU’s relations with Russia but also in its role as a foreign policy actor. Events in Ukraine inevitably dominated the Summit, with EU leaders adopting a balanced approach aimed at achieving three key objectives – de-escalation, containment/deterrence and cooperation – based on political and economic support for Ukraine, increased but limited pressure on Russia, and moves to strengthen ties with other EU neighbours. The Summit also discussed a range of economic and environmental policy issues, with the situation in Ukraine casting a long shadow over the discussion on energy policy, but failed to reach agreement on the EU’s climate goals to 2030, or to put more flesh on the bones of calls for a European “industrial renaissance”. However, two other developments were particularly significant: the creation of the second pillar of the future banking union, establishing a single regime for winding down failing banks; and changes to the savings tax directive, bringing years of negotiation to an end.

Unity stronger than divisions. Ukraine's internal diversity. OSW Point of View Number 40, March 2014

Ukraine is deeply divided internally, although as a result of the changes that have taken place since its independence, the country’s internal divisions now have less and less to do with territorial divides, and the split into historical ‘sub-Ukraines’ has become less pronounced, especially for the younger generation. Ukraine is not a country of two competing regional identities, one in the west, the other in the east. The western identity, in which the unity of Ukraine is a key value, coexists with the multiple and diverse local patriotisms of the different regions in the east and the south of the country. The present protest movement has consolidated the country’s sense of unity. Its opponents have also been championing the indivisibility of Ukraine, even while they demanded a thorough decentralisation of the country, which was often mistaken for separatism. Russia has been stirring up separatist tendencies in Ukraine, but with little success. Crimea is an exception here, because in most respects it has remained unaffected by the dynamics of the social processes transforming mainland Ukraine – separatist tendencies are indeed deeply rooted in the peninsula.

In a state of necessity. How has Orban changed Hungary. OSW Point of View Number 41, April 2014

Viktor Orban’s sweeping victory in the 2010 election ensured his party, Fidesz, a constitutional two-thirds majority in parliament. The party took over the rule of the country from the discredited political left when Hungary was plunged in political and economic crisis. Claiming that the circumstances were unusual and that it had a strong electoral mandate, Fidesz introduced radical changes in the country and thus challenged the previous economic and political order. These changes have led to an unprecedented concentration of power and provoked a discussion on the limits of democracy and the rule of law in the European Union. The state’s economic role has strengthened. The Orban government has been unable to overcome economic stagnation but it has managed to stabilise Hungary’s budget situation, which needs to deal with the high debt. Hungary’s relations with most partners in the EU and NATO have cooled due to controversial moves made by its government. As regards foreign policy and economic co-operation, Orban has granted high priority to the ‘Eastern opening’, where Russia has assumed the leading role.

Security of Russian gas supplies to the EU - the question of infrastrucutral connections. OSW Point of View, February 2005

The energy security of countries importing energy resources depends largely on the shape and quality of operational transport connections. This is particularly important in the case of natural gas supplies. Natural gas is transported mostly by gas pipelines which permanently connect gas producers and consumers. Thus Europe as a consumer is "tied" to certain gas suppliers for anywhere between a dozen and several tens of years. As their own resources are becoming depleted, the EU Member States get increasingly dependent on import of natural gas. The present paper discusses the existing and projected gas transport routes from Russia to the EU. The first part deals with the importance of gas exports to the economy of the Russian Federation, and the second delves into the EU Member States' dependence on gas imports. Then this paper examines the differences in perceiving the energy security issue between the old and the new Member States, those differences stemming from the different degrees of their dependence on Russian supplies. In the third part, two new transport route projects for Russian gas supplies to the EU are compared and it is argued that from the point of view of the Community's interests, the Yamal gas pipeline is a better solution than the North European (Trans-Baltic) gas pipeline.