GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of Gαi subunits

GAIP (G Alpha Interacting Protein) is a member of the recently described RGS (Regulators of G-protein Signaling) family that was isolated by interaction cloning with the heterotrimeric G-protein Gαi3 and was recently shown to be a GTPase-activating protein (GAP). In AtT-20 cells stably expressing GAIP, we found that GAIP is membrane-anchored and faces the cytoplasm, because it was not released by sodium carbonate treatment but was digested by proteinase K. When Cos cells were transiently transfected with GAIP and metabolically labeled with [35S]methionine, two pools of GAIP—a soluble and a membrane-anchored pool—were found. Since the N terminus of GAIP contains a cysteine string motif and cysteine string proteins are heavily palmitoylated, we investigated the possibility that membrane-anchored GAIP might be palmitoylated. We found that after labeling with [3H]palmitic acid, the membrane-anchored pool but not the soluble pool was palmitoylated. In the yeast two-hybrid system, GAIP was found to interact specifically with members of the Gαi subfamily, Gαi1, Gαi2, Gαi3, Gαz, and Gαo, but not with members of other Gα subfamilies, Gαs, Gαq, and Gα12/13. The C terminus of Gαi3 is important for binding because a 10-aa C-terminal truncation and a point mutant of Gαi3 showed significantly diminished interaction. GAIP interacted preferentially with the activated (GTP) form of Gαi3, which is in keeping with its GAP activity. We conclude that GAIP is a membrane-anchored GAP with a cysteine string motif. This motif, present in cysteine string proteins found on synaptic vesicles, pancreatic zymogen granules, and chromaffin granules, suggests GAIP’s possible involvement in membrane trafficking.

Substrate-specific inhibition of RecQ helicase

The RecQ helicases constitute a small but highly conserved helicase family. Proteins in this family are of particular interest because they are critical to maintenance of genomic stability in prokaryotes and eukaryotes. Eukaryotic RecQ helicase family members have been shown to unwind not only DNA duplexes but also DNAs with alternative structures, including structures stabilized by G quartets (G4 DNAs). We report that Escherichia coli RecQ can also unwind G4 DNAs, and that unwinding requires ATP and divalent cation. RecQ helicase is comparably active on duplex and G4 DNA substrates, as measured by direct comparison of protein activity and by competition assays. The porphyrin derivative, N-methyl mesoporphyrin IX (NMM), is a highly specific inhibitor of RecQ unwinding activity on G4 DNA but not duplex DNA: the inhibition constant (Ki) for NMM inhibition of G4 DNA unwinding is 1.7 µM, approximately two orders of magnitude below the Ki for inhibition of duplex DNA unwinding (>100 µM). NMM may therefore prove to be a valuable compound for substrate-specific inhibition of other RecQ family helicases in vitro and in vivo.

Structures of two molluscan hemocyanin genes: Significance for gene evolution

We present here the description of genes coding for molluscan hemocyanins. Two distantly related mollusks, Haliotis tuberculata and Octopus dofleini, were studied. The typical architecture of a molluscan hemocyanin subunit, which is a string of seven or eight globular functional units (FUs, designated a to h, about 50 kDa each), is reflected by the gene organization: a series of eight structurally related coding regions in Haliotis, corresponding to FU-a to FU-h, with seven highly variable linker introns of 174 to 3,198 bp length (all in phase 1). In Octopus seven coding regions (FU-a to FU-g) are found, separated by phase 1 introns varying in length from 100 bp to 910 bp. Both genes exhibit typical signal (export) sequences, and in both cases these are interrupted by an additional intron. Each gene also contains an intron between signal peptide and FU-a and in the 3′ untranslated region. Of special relevance for evolutionary considerations are introns interrupting those regions that encode a discrete functional unit. We found that five of the eight FUs in Haliotis each are encoded by a single exon, whereas FU-f, FU-g, and FU-a are encoded by two, three and four exons, respectively. Similarly, in Octopus four of the FUs each correspond to an uninterrupted exon, whereas FU-b, FU-e, and FU-f each contain a single intron. Although the positioning of the introns between FUs is highly conserved in the two mollusks, the introns within FUs show no relationship either in location nor phase. It is proposed that the introns between FUs were generated as the eight-unit polypeptide evolved from a monomeric precursor, and that the internal introns have been added later. A hypothesis for evolution of the ring-like quaternary structure of molluscan hemocyanins is presented.

Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation.

Central to signaling by fibroblast growth factors (FGFs) is the oligomeric interaction of the growth factor and its high-affinity cell surface receptor, which is mediated by heparin-like polysaccharides. It has been proposed that the binding of heparin-like polysaccharides to FGF induces a conformational change in FGF, resulting in the formation of FGF dimers or oligomers, and this biologically active form is 'presented' to the FGF receptor for signal transduction. In this study, we show that monomeric basic FGF (FGF-2) preferentially self-associates and forms FGF-2 dimers and higher-order oligomers. As a consequence, FGF-2 monomers are oriented for binding to heparin-like polysaccharides. We also show that heparin-like polysaccharides can readily bind to self-associated FGF-2 without causing a conformational change in FGF-2 or disrupting the FGF-2 self-association, but that the bound polysaccharides only additionally stabilize the FGF-2 self-association. The preferential self-association corresponds to FGF-2 translations along two of the unit cell axes of the FGF-2 crystal structures. These two axes represent the two possible heparin binding directions, whereas the receptor binding sites are oriented along the third axis. Thus, we propose that preferential FGF-2 self-association, further stabilized by heparin, like "beads on a string," mediates FGF-2-induced receptor dimerization and activation. The observed FGF-2 self-association, modulated by heparin, not only provides a mechanism of growth factor activation but also represents a regulatory mechanism governing FGF-2 biological activity.

Training and search methods for speech recognition.

Speech recognition involves three processes: extraction of acoustic indices from the speech signal, estimation of the probability that the observed index string was caused by a hypothesized utterance segment, and determination of the recognized utterance via a search among hypothesized alternatives. This paper is not concerned with the first process. Estimation of the probability of an index string involves a model of index production by any given utterance segment (e.g., a word). Hidden Markov models (HMMs) are used for this purpose [Makhoul, J. & Schwartz, R. (1995) Proc. Natl. Acad. Sci. USA 92, 9956-9963]. Their parameters are state transition probabilities and output probability distributions associated with the transitions. The Baum algorithm that obtains the values of these parameters from speech data via their successive reestimation will be described in this paper. The recognizer wishes to find the most probable utterance that could have caused the observed acoustic index string. That probability is the product of two factors: the probability that the utterance will produce the string and the probability that the speaker will wish to produce the utterance (the language model probability). Even if the vocabulary size is moderate, it is impossible to search for the utterance exhaustively. One practical algorithm is described [Viterbi, A. J. (1967) IEEE Trans. Inf. Theory IT-13, 260-267] that, given the index string, has a high likelihood of finding the most probable utterance.

On the mechanism of skin wound "contraction": a granulation tissue "knockout" with a normal phenotype.

This report explores the mechanism of spontaneous closure of full-thickness skin wounds. The domestic pig, often used as a human analogue for skin wound repair studies, closes these wounds with kinetics similar to those in the guinea pig (mobile skin), even though the porcine dermis on the back is thick and nearly immobile. In the domestic pig, as in the guinea pig, daily full-thickness excisions of the central granulation tissue up to but not including the wound edges in both back and flank wounds do not alter the rate or completeness of wound closure or the final pattern of the scar. A purse-string mechanism of closure was precluded by showing that surgical interruption of wound edge continuity does not alter closure kinetics or wound shape. We conclude that "tightness" of skin is not a key factor nor is the central granulation tissue required for normal wound closure. These data imply that in vitro models such as contraction of isolated granulation tissue or of the cell-populated collagen lattice may not be relevant for understanding the cell biology of in vivo wound closure. Implications for the mechanism for wound closure are discussed.

Redistribution of synaptic vesicles and their proteins in temperature-sensitive shibire(ts1) mutant Drosophila.

From an extract of Drosophila melanogaster head homogenates, a membrane fraction can be isolated that has the same sedimentation properties as vertebrate synaptic vesicles and contains Drosophila synaptotagmin. The fraction disappears from homogenates of temperature-sensitive (ts) mutant shibire(ts1) (shi(ts1)) flies paralyzed by exposure to non-permissive temperatures, and reappears on return to permissive temperatures. Since reversible, temperature-dependent depletion of synaptic vesicles is known to occur in shibire(ts1) flies, we conclude that the fraction we have identified contains synaptic vesicles. We have examined the fate of synaptic vesicle membrane proteins in shibire flies at nonpermissive temperatures and found that all of these vesicle antigens are transferred to rapidly sedimenting membranes and codistribute with a plasma membrane marker by both glycerol velocity and metrizamide density sedimentation and by confocal microscopy. Three criteria were used to establish that other neuron-specific antigens--neuronal synaptobrevin and cysteine-string proteins--are legitimate components of synaptic vesicles: cosedimentation with Drosophila synaptotagmin, immunoadsorption, and disappearance of these antigens from the vesicle fractions in paralyzed shibire flies.

Reutilização de baterias automotivas como fonte alternativa de energia.

Atualmente um dos principais objetivos na área de pesquisa tecnológica é o desenvolvimento de soluções em favor do Meio Ambiente. Este trabalho tem por objetivo demonstrar a reutilização e consequentemente o aumento da vida útil de uma bateria Chumbo-Ácido, comumente instaladas em veículos automóveis, bem como beneficiar locais e usuários remotos onde o investimento na instalação de linhas de transmissão se torna inviável geográfica e economicamente, utilizando a luz solar como fonte de energia. No entanto a parte mais suscetível a falhas são as próprias baterias, justamente pela vida útil delas serem pequenas (em torno de 3 anos para a bateria automotiva) em comparação com o restante do sistema. Considerando uma unidade que já foi usada anteriormente, a possibilidade de falhas é ainda maior. A fim de diagnosticar e evitar que uma simples bateria possa prejudicar o funcionamento do sistema como um todo, o projeto considera a geração de energia elétrica por células fotovoltaicas e também contempla um sistema microcontrolado para leitura de dados utilizando o microcontrolador ATmega/Arduino, leitura de corrente por sensores de efeito hall da Allegro Systems, relés nas baterias para abertura e fechamento delas no circuito e um sistema de alerta para o usuário final de qual bateria está em falha e que precisa ser reparada e/ou trocada. Esse projeto foi montado na Ilha dos Arvoredos SP, distante da costa continental em aproximadamente 2,0km. Foram instaladas células solares e um banco de baterias, a fim de estudar o comportamento das baterias. O programa pôde diagnosticar e isolar uma das baterias que estava apresentando defeito, a fim de se evitar que a mesma viesse a prejudicar o sistema como um todo. Por conta da dificuldade de locomoção imposta pela geografia, foi escolhido o cartão SD para o armazenamento dos dados obtidos pelo Arduino. Posteriormente os dados foram compilados e analisados. A partir dos resultados apresentados podemos concluir que é possível usar baterias novas e baterias usadas em um mesmo sistema, de tal forma que se alguma das baterias apresentar uma falha o sistema por si só isolará a unidade.

Sangre fresca publicitaria: True Blood y las transfusiones de la ficción

El artículo analiza el concepto de transmedia storytelling en su dimensión publicitaria. En cierto modo, las narrativas transmediáticas, esto es, la diseminación de las tramas ficcionales a lo largo de soportes y plataformas diversos pero sinérgicos (letra impresa, cine, televisión) fueron empleadas en primer lugar en el mensaje publicitario, obligado a transmigrar por su carencia de un soporte exclusivo y a cambio por la vocación de colonizarlos a todos. Hoy día es el producto cultural ficcional el que devuelve a la publicidad el préstamo: son los universos ficcionales los marcos más propiciatorios para el consumo, no sólo de la cultura industrial en sí, en cualquiera de sus soportes, sino de todo un rosario de marcas arracimadas en torno a la narrativa ficcional. Analizaremos en detalle este fenómeno en una teleserie norteamericana muy reciente, True Blood (HBO, 2008), todavía en emisión, y en todo el universo ficcional que orbita a su alrededor. La diferencia entre los productos culturales en sí y las piezas publicitarias encargadas de promocionarlos –como ya vio premonitoriamente Adorno- se disuelve en un continuo cross-promotional, que tiende a enmascarar también los límites entre las estrategias corporativas y las intervenciones del fan.

On the existence of fractal strings whose set of dimensions of fractality is not perfect

In this paper we give an example of a nonlattice self-similar fractal string such that the set of real parts of their complex dimensions has an isolated point. This proves that, in general, the set of dimensions of fractality of a fractal string is not a perfect set.

On the existence of exponential polynomials with prefixed gaps

This paper shows that the conjecture of Lapidus and Van Frankenhuysen on the set of dimensions of fractality associated with a nonlattice fractal string is true in the important special case of a generic nonlattice self-similar string, but in general is false. The proof and the counterexample of this have been given by virtue of a result on exponential polynomials P(z), with real frequencies linearly independent over the rationals, that establishes a bound for the number of gaps of RP, the closure of the set of the real projections of its zeros, and the reason for which these gaps are produced.

" Adoremus in æternum" &c: a canon for three tenors or sopranos

by Louis Cherubini ; with an accompt for stringed instts or pianoforte ad lib.

Latin exercise book of James Baker, 1759-1760

Small notebook kept by James Baker in the late 1750s; the dates 1755, 1756, and 1758 were written in the book. The volume contains Latin theses, Latin translations from the Book of Genesis, and three pages of English text recording an argument about the soul. The notebook has a string binding and pages of different size. The text does not appear to follow a system of organization and includes scribbles and struck-out words.

[ Pûtlā nav ćhûgā

Pīr Satgur Nūr].

Prevalência de lesões musculo-esqueléticas em profissionais de artes musicais

Enquadramento: As lesões músculo-esqueléticas relacionadas com o trabalho apresentam-se atualmente como um crescente flagelo e a profissão de músico é uma atividade predisponente para a ocorrência dessas lesões, exigindo uma atenção especial dos profissionais de saúde para identificar e controlar os fatores de risco. Objetivos: O presente estudo pretende identificar a prevalência das perturbações músculo-esqueléticas nos músicos profissionais praticantes de Cordofones beliscados e analisar a sua relação com as variáveis sociodemográficas, estado de saúde e características da prática instrumental. Métodos: Trata-se de um estudo não experimental, transversal, descritivo-correlacional e de caráter quantitativo, que envolveu 70 músicos portugueses praticantes de cordofones beliscados, residentes em Viseu, Porto e Lisboa, com idades compreendidas entre 18 e 55 anos. Foi realizado com recurso ao uso de um questionário que avalia as variáveis sociodemográficas, as clínicas e as relacionadas com a prática musical e para avaliação das perturbações musculoesqueléticas utilizámos o “Questionário Nórdico Músculo-Esquelético”. Resultados: Dos músicos estudados 70,0% referem sentir perturbações músculo-esqueléticas como dor/formigueiro/dormência nos últimos 12 meses, tendo estes ocorrido sobretudo nos punhos/mãos (68.6%), ombros (54.3%), pescoço e região lombar (44.3%). Observámos que são vários os fatores risco das perturbações musculoesqueléticas como a idade avançada, um Índice de Massa Corporal mais elevado, o consumo de bebidas alcoólicas, não praticar atividade física, o excesso de espetáculos, a postura adotada, instrumentos mais pesados e tocar sem pausas. Conclusões: O nosso estudo reforça a ideia de que as perturbações músculo-esqueléticas estão presentes nos músicos portugueses e que a sua origem está relacionada com características individuais, estado de saúde e exigências da prática instrumental. Cabe então ao Enfermeiro de Reabilitação criar estratégias preventivas, para evitar que os fatores de risco acrescidos causem ou aumentem as já instaladas lesões músculo-esqueléticas nos músicos, promovendo uma carreira longa e saudável. Palavras-chave: Músicos; Lesões musculoesqueléticas; Fatores de risco; Enfermagem de Reabilitação.