A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin

During light-driven proton transport bacteriorhodopsin shuttles between two protein conformations. A large-scale structural change similar to that in the photochemical cycle is produced in the D85N mutant upon raising the pH, even without illumination. We report here that (i) the pKa values for the change in crystallographic parameters and for deprotonation of the retinal Schiff base are the same, (ii) the retinal isomeric configuration is nearly unaffected by the protein conformation, and (iii) preventing rotation of the C13—C14 double bond by replacing the retinal with an all-trans locked analogue makes little difference to the Schiff base pKa. We conclude that the direct cause of the conformational shift is destabilization of the structure upon loss of interaction of the positively charged Schiff base with anionic residues that form its counter-ion.

Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers

For the act of membrane fusion, there are two competing, mutually exclusive molecular models that differ in the structure of the initial pore, the pathway for ionic continuity between formerly separated volumes. Because biological “fusion pores” can be as small as ionic channels or gap junctions, one model posits a proteinaceous initial fusion pore. Because biological fusion pore conductance varies widely, another model proposes a lipidic initial pore. We have found pore opening and flickering during the fusion of protein-free phospholipid vesicles with planar phospholipid bilayers. Fusion pore formation appears to follow the coalescence of contacting monolayers to create a zone of hemifusion where continuity between the two adherent membranes is lipidic, but not aqueous. Hypotonic stress, causing tension in the vesicle membrane, promotes complete fusion. Pores closed soon after opening (flickering), and the distribution of fusion pore conductance appears similar to the distribution of initial fusion pores in biological fusion. Because small flickering pores can form in the absence of protein, the existence of small pores in biological fusion cannot be an argument in support of models based on proteinaceous pores. Rather, these results support the model of a lipidic fusion pore developing within a hemifused contact site.

Role of adenine functional groups in the recognition of the 3′-splice-site AG during the second step of pre-mRNA splicing

The AG dinucleotide at the 3′ splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG → GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG → CG) or uracil (AG → UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG → HG). When adenine was replaced by a purine base (AG → PG) or by 7-deazaadenine (AG → c7AG), little effect on the second step was observed, suggesting that the 6-NH2 and N7 groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG → 2-APG) had no effect on the second step. Taken together, our results suggest that the N1 group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.

Type 2 diabetes: Evidence for linkage on chromosome 20 in 716 Finnish affected sib pairs

We are conducting a genome scan at an average resolution of 10 centimorgans (cM) for type 2 diabetes susceptibility genes in 716 affected sib pairs from 477 Finnish families. To date, our best evidence for linkage is on chromosome 20 with potentially separable peaks located on both the long and short arms. The unweighted multipoint maximum logarithm of odds score (MLS) was 3.08 on 20p (location, x̂ = 19.5 cM) under an additive model, whereas the weighted MLS was 2.06 on 20q (x̂ = 57 cM, recurrence risk, λ̂s = 1.25, P = 0.009). Weighted logarithm of odds scores of 2.00 (x̂ = 69.5 cM, P = 0.010) and 1.92 (x̂ = 18.5 cM, P = 0.013) were also observed. Ordered subset analyses based on sibships with extreme mean values of diabetes-related quantitative traits yielded sets of families who contributed disproportionately to the peaks. Two-hour glucose levels in offspring of diabetic individuals gave a MLS of 2.12 (P = 0.0018) at 9.5 cM. Evidence from this and other studies suggests at least two diabetes-susceptibility genes on chromosome 20. We have also screened the gene for maturity-onset diabetes of the young 1, hepatic nuclear factor 4-a (HNF-4α) in 64 affected sibships with evidence for high chromosomal sharing at its location on chromosome 20q. We found no evidence that sequence changes in this gene accounted for the linkage results we observed.

Statistical significance of protein structure prediction by threading

In this study, we estimate the statistical significance of structure prediction by threading. We introduce a single parameter ɛ that serves as a universal measure determining the probability that the best alignment is indeed a native-like analog. Parameter ɛ takes into account both length and composition of the query sequence and the number of decoys in threading simulation. It can be computed directly from the query sequence and potential of interactions, eliminating the need for sequence reshuffling and realignment. Although our theoretical analysis is general, here we compare its predictions with the results of gapless threading. Finally we estimate the number of decoys from which the native structure can be found by existing potentials of interactions. We discuss how this analysis can be extended to determine the optimal gap penalties for any sequence-structure alignment (threading) method, thus optimizing it to maximum possible performance.

Inhibition of caspase 1 reduces human myocardial ischemic dysfunction via inhibition of IL-18 and IL-1β

The proinflammatory cytokine IL-18 was investigated for its role in human myocardial function. An ischemia/reperfusion (I/R) model of suprafused human atrial myocardium was used to assess myocardial contractile force. Addition of IL-18 binding protein (IL-18BP), the constitutive inhibitor of IL-18 activity, to the perifusate during and after I/R resulted in improved contractile function after I/R from 35% of control to 76% with IL-18BP. IL-18BP treatment also preserved intracellular tissue creatine kinase levels (by 420%). Steady-state mRNA levels for IL-18 were elevated after I/R, and the concentration of IL-18 in myocardial homogenates was increased (control, 5.8 pg/mg vs. I/R, 26 pg/mg; P < 0.01). Active IL-18 requires cleavage of its precursor form by the IL-1β-converting enzyme (caspase 1); inhibition of caspase 1 also attenuated the depression in contractile force after I/R (from 35% of control to 75.8% in treated atrial muscle; P < 0.01). Because caspase 1 also cleaves the precursor IL-1β, IL-1 receptor blockade was accomplished by using the IL-1 receptor antagonist. IL-1 receptor antagonist added to the perifusate also resulted in a reduction of ischemia-induced contractile dysfunction. These studies demonstrate that endogenous IL-18 and IL-1β play a significant role in I/R-induced human myocardial injury and that inhibition of caspase 1 reduces the processing of endogenous precursors of IL-18 and IL-1β and thereby prevents ischemia-induced myocardial dysfunction.

Site-specific mutations in the mature form of human IL-18 with enhanced biological activity and decreased neutralization by IL-18 binding protein

IL-18 can be considered a proinflammatory cytokine mediating disease as well as an immunostimulatory cytokine that is important for host defense against infection and cancer. The high-affinity, constitutively expressed, and circulating IL-18 binding protein (IL-18BP), which competes with cell surface receptors for IL-18 and neutralizes IL-18 activity, may act as a natural antiinflammatory as well as immunosuppressive molecule. In the present studies, the IL-18 precursor caspase-1 cleavage site was changed to a factor Xa site, and, after expression in Escherichia coli, mature IL-18 was generated by factor Xa cleavage. Mature IL-18 generated by factor Xa cleavage was fully active. Single point mutations in the mature IL-18 peptide were made, and the biological activities of the wild-type (WT) IL-18 were compared with those of the mutants. Mutants E42A and K89A exhibited 2-fold increased activity compared with WT IL-18. A double mutant, E42A plus K89A, exhibited 4-fold greater activity. Unexpectedly, IL-18BP failed to neutralize the double mutant E42A plus K89A compared with WT IL-18. The K89A mutant was intermediate in being neutralized by IL-18BP, whereas neutralization of the E42A mutant was comparable to that in the WT IL-18. The identification of E42 and K89 in the mature IL-18 peptide is consistent with previous modeling studies of IL-18 binding to IL-18BP and explains the unusually high affinity of IL-18BP for IL-18.

Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels

Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K+ (KATP) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with KATP channel-dependent membrane excitability remains elusive. Here, we identify that the response of KATP channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the KATP channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and KATP channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with KATP channels and associated functions.

2002 Parliamentary elections in Ukraine: Events, results and consequences; Ukraine's Parliamentary system after the elections. OSW Study 8/2003

2002 elections: On 31 March 2002, parliamentary elections were held in Ukraine. As expected, they were a major success for the centrist-rightist coalition focused around former Prime Minister Viktor Yuschenko. The communists emerged significantly weaker from the vote, and the "party of power" achieved a poor result. Yet, due to the mixed electoral law (half of the deputies were elected in single-mandate districts), the latter block, firmly supported by President Leonid Kuchma, resulted as the main force in Parliament. The results of particular parties and blocks were as follows: Viktor Yuschenko's Block received 23.57% of votes and 112 seats, the Communist Party of Ukraine - 19.98% of votes and 66 seats, the "For One Ukraine" block - 11.77% of votes and 101 seats, Yulia Tymoshenko's Block - 7.26% of votes and 22 seats, the Socialist Party of Ukraine - 6.87% of votes and 22 seats, and the Social Democratic Party of Ukraine (united) - 6.27% of votes and 24 seats. This shows how the mixed electoral regulations favour "For One Ukraine" and act against Yuschenko's block. One should note, however, that the latter gained the support of less than one quarter of voters. After the election: The dominant force in Ukraine's Verkhovna Rada, elected in March 2002, are the deputies of "One Ukraine", a fraction of the pro-presidential centre. "One Ukraine" has refused to admit any of the opposition's representatives (either from the right or left wings) into the parliament's presidium, but has accepted opposition-appointed heads of many parliamentary commissions. Viktor Yuschenko's "Our Ukraine", which has been the largest parliamentary fraction since June, attempted to proclaim itself the centre of the parliamentary majority, but its policy was awkward and inconsistent, and the main success of this club was that it didn't break up. Viktor Yuschenko's moves have been particularly incoherent and they undermined the image of Yuschenko as Ukraine's future leader, created throughout the course of the electoral campaign. In autumn, the main oligarchic groups and their representative fractions ("One Ukraine", which proved to be a useless instrument, was dissolved in June), reached a compromise with the president. It was agreed that the new prime minister should be a Donetsk clan representative (Viktor Yanukovych), and that the Dnipropetrovsk clan should appoint the president of the National Bank of Ukraine (this position went to Serhij Tihipko). The Kyiv clan obtained the President's Administration (Viktor Medvedchuk was appointed in spring) and a considerable number of parliamentary commissions. The president's interests in the government are to be protected by Mykola Azarov, former Head of the State Tax Administration. This compromise "package" was designed to secure the shares of the main oligarchic clans in the power and the president's strong position as mediator.

The oligarchic democracy: The influence of business groups on Ukrainian politics. OSW Study 42/2012

The Ukrainian oligarchic system, which developed into its ultimate shape during Leonid Kuchma’s second presidency, turned out to be very durable. The nature of close relations between the government and the oligarchs has not undergone any major changes either as a consequence of the Orange Revolution or following Victor Yanukovych’s victory in the presidential election of 2010. Although reshuffles have taken place inside the political and business elites, nothing seems to be able to change this system, at least in the medium term. This text is aimed at presenting the network of connections existing between big business and politics in Ukraine and at pointing to the key oligarchic groups and the political forces they support. A definite majority of papers concerning contemporary Ukrainian politics as a rule disregard or deal with this subject very superficially, while it is impossible to understand modern Ukraine without understanding a number of dependencies existing between the political and business elites there.

Proposta di sottotitolaggio del terzo episodio del film Operacija Y i drugie priključenija Šurika [Operazione Y e altre avventure di Šurik]

L’obiettivo del presente elaborato è quello di fornire un testo per il sottotitolaggio di Operacija Y [Operazione Y], il terzo episodio della celebre commedia sovietica Operacija Y i drugie priključenija Šurika [Operazione Y e altre avventure di Šurik]. Il capitolo iniziale introduce la traduzione audiovisiva e tratta in particolare il sottotitolaggio, i passaggi di realizzazione di quest’ultimo e i suoi tratti salienti. Il secondo capitolo propone una contestualizzazione del genere della commedia cinematografica russa durante il periodo sovietico, mentre il terzo fornisce una breve biografia del regista Leonid Gajdaj e la descrizione del film, con particolare attenzione al rinomato trio comico criminale formato da Trus, Balbes e Byvalyj. L’ultimo capitolo consiste in un commento alla traduzione e nell’analisi delle difficoltà incontrate e delle soluzioni trovate. In appendice viene riportato il testo per la sottotitolazione.

Proceedings of the 4th International Conference on Applied Innovations in IT

This volume contains publications of the International Conference on Applied Innovations in IT (ICAIIT), which took place in Koethen March 10th 2016. The conference is devoted to problems of applied research in the fields of automation and communications. The research results can be of interest for researchers and development engineers, who deal with theoretical base and the application of the knowledge in the respective areas.

Possible Approaches to Commercializing Applied University Research: a Russian Case Study

This paper discusses the environment around universities in Russia and possible approaches to analyzing and choosing the method for applied research results commercialization as well as selecting promising applied research areas in that environment. Conceptual foundations for decision making during the commercialization and roadmap/action plan creation processes are outlined. These can be useful to both universities for planning their activities aswell as for organizations that plan to cooperate with universities or that are interested in university generated research. This being said, obtained models and used evaluation parameters may be unique and may depend upon the particular project, university, region, and personal preferences of decision makers. Thus, consideration of these parameters and characteristics only has merit when making decisions in the dynamics of change of these parameters. For this purpose statistical information is needed that characterizes the competencies of the research organization (university) inquestion, needs of partner organizations, governmental and societal requirements, and science and technology prospects. After determining the promising research areas it’s time to look at particular projects, which in turn are also characterized by various parameters dependent upon their objectives. Considering the values of these parameters in their dynamics allows control of project parameters in the course of its execution. This in turn allows prediction of negative situations and alleviation of such by setting the target values of parameters and using best practices and standardization of management processes to achieve those values.

Radiocarbon ages from Novaya Zemlya, Russia

Recent observations on postglacial emergence and past glacier extent for one of the least accessible areas in the Arctic, northern Novaya Zemlya are here united. The postglacial marine limit formed 5 to 6 ka is registered on the east and west coasts of the north island at 10 ± 1 and 18 ± 2 m aht, respectively. This modest and late isostatic response along with deglacial ages of >9.2 ka on adjacent marine cores from the northern Barents Sea indicate either early (>13 ka) deglaciation or modest ice sheet loading (<1500 m thick ice sheet) of Novaya Zemlya. Older and higher (up to 50 m aht) raised beaches were identified beneath a discontinuous glacial drift. Shells from the drift and underlying sublittoral sediments yield minimum limiting 14C ages of 26 to 30 ka on an earlier deglacial event(s). The only moraines identified are within 4 km of present glacier margins and reflect at least three neoglacial advances in the past 2.4 ka.