A new firefly luciferase with bimodal spectrum: Identification of structural determinants of spectral pH-sensitivity in firefly luciferases

Fireflies emit flashes in the green-yellow region of the spectrum for the purpose of sexual attraction. The bioluminescence color is determined by the luciferases. It is well known that the in vitro bioluminescence color of firefly luciferases can be shifted toward the red by lower pH and higher temperature; for this reason they are classified as pH-sensitive luciferases. However, the mechanism and structural origin of pH sensitivity in fireflies remains unknown. Here we report the cloning of a new luciferase from the Brazilian twilight active firefly Macrolampis sp2, which displays an unusual bimodal spectrum. The recombinant luciferase displays a sensitive spectrum with the peak at 569 nm and a shoulder in the red region. Comparison of the bioluminescence spectra of Macrolampis, Photinus and Cratomorphus firefly luciferases shows that the distinct colors are determined by the ratio between green and red emitters under luciferase influence. Comparison of Macrolampis luciferase with the highly similar North American Photinus pyralis luciferase (91%) showed few substitutions potentially involved with the higher spectral sensitivity in Macrolampis luciferase. Site-directed mutagenesis showed that the natural substitution E354N determines the appearance of the shoulder in the red region of Macrolampis luciferase bioluminescence spectrum, helping to identify important interactions and residues involved in the pH-sensing mechanism in firefly luciferases. © 2005 American Society for Photobiology.

Bovine serum albumin displays luciferase-like activity in presence of luciferyl adenylate: Insights on the origin of protoluciferase activity and bioluminescence colours

Luciferyl adenylate, the key intermediate in beetle bioluminescence, is produced through adenylation of D-luciferin by beetle luciferases and also by mealworm luciferase-like enzymes which produce a weak red chemiluminescence. However, luciferyl adenylate is only weakly chemiluminescent in water at physiological pH and it is unclear how efficient bioluminescence evolved from its weak chemiluminescent properties. We found that bovine serum albumin (BSA) and neutral detergents enhance luciferyl adenylate chemiluminescence by three orders of magnitude, simulating the mealworm luciferase-like enzyme chemiluminescence properties. These results suggest that the beetle protoluciferase activity arose as an enhanced luciferyl adenylate chemiluminescence in the protein environment of the ancestral AMP-ligase. The predominance of luciferyl adenylate chemiluminescence in the red region under most conditions suggests that red luminescence is a more primitive condition that characterized the original stages of protobioluminescence, whereas yellow-green bioluminescence may have evolved later through the development of a more structured and hydrophobic active site. Copyright © 2006 John Wiley & Sons, Ltd.

Few substitutions affect the bioluminescence spectra of Phrixotrix (Coleoptera: Phengodidae) luciferases: a site-directed mutagenesis survey

Phrixotrix (railroad worm) luciferases produce bioluminescence in the green and red regions of the spectrum, depending on the location of the lanterns, and are the only luciferases naturally producing red bioluminescence. Comparison of the luciferase sequences showed a set of substitutions that could be involved in bioluminescence colour determination: (a) unique substitutions in the red luciferase replacing otherwise invariant residues; (b) conserved basic residues in the green-yellow emitting luciferases; and (c) an additional R353 residue in red-emitting luciferase (Viviani et al., 1999). To investigate whether these sites have a functional role in bioluminescence colour determination, we performed a site-directed mutagenesis. Natural substitutions in the region 220-344 and residues in the putative luciferin-binding site were also investigated. With the exception of the previously identified substitution of R215 and T226 (Viviani et al., 2002), which display dramatic red-shift effects on the spectrum of green-yellow-emitting luciferases, only a few substitutions had a moderate effect on the spectrum of the green-emitting luciferase. In contrast, no single substitution affected the spectrum of the red-emitting luciferase. The results suggest that the identity of the active site residues is not so critical for determining red bioluminescence in PxRE luciferase. Rather, the conformation assumed during the emitting step could be critical to set up proper interactions with excited oxyluciferin. Copyright ©2007 John Wiley & Sons, Ltd.

The influence of the loop between residues 223-235 in beetle luciferase bioluminescence spectra: A solvent gate for the active site of pH-sensitive luciferases

Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red. Firefly luciferases can shift the spectrum to red in response to pH and temperature changes, whereas click beetle and railroadworm luciferases do not. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. Through comparative site-directed mutagenesis and modeling studies, using the pH-sensitive luciferases (Macrolampis and Cratomorphus distinctus fireflies) and the pH-insensitive luciferases (Pyrearinus termitilluminans, Phrixotrix viviani and Phrixotrix hirtus) cloned by our group, here we show that substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). The substitutions at positions 227, 228 and 229 (P. pyralis sequence) cause dramatic redshift and temporal shift in both groups of luciferases, indicating their involvement in labile interactions. Modeling studies showed that the residues Y227 and N229 are buried in the protein core, fixing the loop to other structural elements participating at the bottom of the luciferin binding site. Changes in pH and temperature (in firefly luciferases), as well as point mutations in this loop, may disrupt the interactions of these structural elements exposing the active site and modulating bioluminescence colors. © 2007 The Authors.

Luciferase de Macrolampis SP como gene repórter dual em biosensores simultâneos de expressão gênica e variações de intracelulares de ph, concentrações de fosfato e cátions divalentes de metais pesados

Trata-se a presente invenção de um produto da área de biotecnologia constituído de Luciferase de Microlampis SP recombinante apresenta espectro de bioluminescência bimodal e sensibilidade elevada a mudanças de pH- e temperatura, como gene repórter dual que codifica uma proteína bioluminosa para uso em biosensores simultâneos de expressão gênica e variações de intracelulares de PH, com concentrações de Fosfato e Cátions divalentes de metais pesados, e presença de ATP e como biosensor intracelular de mudanças de pH e outras condições homeostáticas.

Thiophenecarboxamide derivatives activated by EthA kill mycobacterium tuberculosis by inhibiting the CTP synthetase PyrG

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

On Equilibrium Distribution of a Reversible Growth Model

We study a probabilistic model of interacting spins indexed by elements of a finite subset of the d-dimensional integer lattice, da parts per thousand yen1. Conditions of time reversibility are examined. It is shown that the model equilibrium distribution converges to a limit distribution as the indexing set expands to the whole lattice. The occupied site percolation problem is solved for the limit distribution. Two models with similar dynamics are also discussed.

Proposta di sottotitolazione in italiano del film "Я шагаю по Москве" (1963)

Il tema del mio elaborato finale è la sottotitolazione del film “A passeggio per Mosca” di Georgij Danelija (1963), un noto regista russo. I suoi film più famosi sono “Мимино” (Mimino) e “Осенный марафон” (“Maratona d’autunno”). L’attore protagonista del film è Nikita Michalkov, famoso attore e regista, nonché personaggio controverso della vita culturale russa; il suo film più famoso è “Утомлённые солнцем” (“Sole ingannatore”). I personaggi più anziani furono interpretati da attori famosi quali Rolan Bykov, attore di teatro, e Vladimir Basov, regista e attore straordinariamente versatile. I ruoli principali, invece, furono affidati ad attori meno noti: Aleksej Loktev e Evgenij Steblov, che si dedicarono entrambi al teatro per gran parte della propria vita, e Galina Pol’skich, che divenne una grande attrice di film drammatici. Straordinari sono anche il lavoro dell’operatore cinematografico Vadim Jusov e quello del compositore Andrej Petrov. Il film suscitò diverse impressioni nel pubblico e nella critica e fu accolto positivamente da Chruščёv: presto sarebbe diventato un simbolo del “Disgelo”, un periodo in cui Chruščёv effettuò un’opera di destalinizzazione dell’URSS: il clima era quindi più disteso, sebbene vi fosse un malcontento diffuso a causa dell’economia fallimentare dello Stato. La canzone finale, che contribuì al successo del film, esprime perfettamente quest’atmosfera spensierata, che pervade anche Mosca, la vera protagonista del film. Della capitale vengono inquadrati i luoghi più importanti: tra questi il parco Gorkij, la metropolitana e il GUM. Tradurre i sottotitoli del film non è stato facile. In particolare, ho incontrato difficoltà nel tradurre alcune particelle russe che non hanno un equivalente in italiano e battute in cui erano presenti importanti riferimenti culturali. Il lavoro a cui mi sono dedicata con più impegno è stato la traduzione della canzone finale: desideravo che i sottotitoli italiani fossero in rima e conservassero il significato dei versi originari.

Creation of Computer Simulation Model for Ecological Consequences Prognosis of Toxic Chemicals Discharge from Sunken on Baltic Sea Bottom Weapons"

This study summarises all the accessible data on old German chemical weapons dumped in the Baltic Sea. Mr. Goncharov formulated a concept of ecological impact evaluation of chemical warfare agents (CWA) on the marine environment and structured a simulation model adapted to the specific character of the hydrological condition and hydrobiological subjects of the Bornholm Deep. The mathematical model he has created describes the spreading of contaminants by currents and turbulence in the near bottom boundary layer. Parameters of CWA discharge through corrosion of canisters were given for various kinds of bottom sediments with allowance for current velocity. He created a method for integral estimations and a computer simulation model and completed a forecast for CWA "Mustard", which showed that in normal hydrometeorological conditions there are local toxic plumes drifting along the bottom for a distance of up to several kilometres. With storm winds the toxic plumes from separate canisters interflow and lengthen and can reach fishery areas near Bornholm Island. When salt water from the North Sea flows in, the length of toxic zones can increase up to and over 100 kilometres and toxic water masses can spread into the northern Baltic. On this basis, Mr. Goncharov drew up recommendations to reduce dangers for human ecology and proposed the creation of a special system for the forecasting and remote sensing of the environmental conditions of CWA burial places.

The Ingrian Question as a Historical and Political Phenomenon

The project dealt with the political history of the Finnish-speaking minorities of the Russian northwest, mainly in the 20th century. The first part looks at the development of the national movement of the Ingrian Finns and other related ethnic groups (Izhoras, Votes) from the turn of the century to 1920, when Estonia and Finland signed peace treaties with Soviet Russia and the national rights of the Finnish minority in Russia were to some extent guaranteed. In the second section, on the history of the Ingrians during Soviet and post-Soviet times, areas covered include Ingrian national-cultural autonomy in the 1920s, the activities of Ingrian "ingri" organizations in Finland during the inter-war period, social and national repression and the end of autonomy in the 1930s, the dispersal of the Ingrians during the second world war, their first attempts to return home in the immediate post-war period, trends in the development of the social and cultural life of Ingrians during the last 40 years, and the prospects for their existence as an ethnic unity in the future. The research is based on documentary sources from 15 Russian archives, many of which have not previously been used.

FUNCTIONAL PRINCIPAL COMPONENTS MODEL FOR HIGH-DIMENSIONAL BRAIN IMAGING

We establish a fundamental equivalence between singular value decomposition (SVD) and functional principal components analysis (FPCA) models. The constructive relationship allows to deploy the numerical efficiency of SVD to fully estimate the components of FPCA, even for extremely high-dimensional functional objects, such as brain images. As an example, a functional mixed effect model is fitted to high-resolution morphometric (RAVENS) images. The main directions of morphometric variation in brain volumes are identified and discussed.

Isospin breaking in pion–deuteron scattering and the pion–nucleon scattering lengths

In recent years, high-accuracy data for pionic hydrogen and deuterium have become the primary source of information on the pion–nucleon scattering lengths. Matching the experimental precision requires, in particular, the study of isospin-breaking corrections both in pion– nucleon and pion–deuteron scattering. We review the mechanisms that lead to the cancellation of potentially enhanced virtual-photon corrections in the pion–deuteron system, and discuss the subtleties regarding the definition of the pion–nucleon scattering lengths in the presence of electromagnetic interactions by comparing to nucleon–nucleon scattering. Based on the p±p channels we find for the virtual-photon-subtracted scattering lengths in the isospin basis a1/2/ g= (170.5±2.0) · 10−3M−1p and a3/2/ g= (−86.5±1.8) · 10−3M−1p .