Cryo-electron microscopy studies of empty capsids of human parvovirus B19 complexed with its cellular receptor.

The three-dimensional structures of human parvovirus B19 VP2 capsids, alone and complexed with its cellular receptor, globoside, have been determined to 26 resolution. The B19 capsid structure, reconstructed from cryo-electron micrographs of vitrified specimens, has depressions on the icosahedral 2-fold and 3-fold axes, as well as a canyon-like region around the 5-fold axes. Similar results had previously been found in an 8 angstrom resolution map derived from x-ray diffraction data. Other parvoviral structures have a cylindrical channel along the 5-fold icosahedral axes, whereas density covers the 5-fold axes in B19. The glycolipid receptor molecules bind into the depressions on the 3-fold axes of the B19:globoside complex. A model of the tetrasaccharide component of globoside, organized as a trimeric fiber, fits well into the difference density representing the globoside receptor. Escape mutations to neutralizing antibodies map onto th capsid surface at regions immediately surrounding the globoside attachment sites. The proximity of the antigenic epitopes to the receptor site suggests that neutralization of virus infectivity is caused by preventing attachment of viruses to cells.

Purification of the Drosophila RNA polymerase II general transcription factors.

We describe a fractionation and purification scheme for the Drosophila RNA polymerase II general transcription factors. Drosophila TFIIE, TFIIF, TFIIH, and RNA polymerase II have been purified to greater than 50% homogeneity from Drosophila embryo nuclear extracts. TFIID has been purified 80-fold and is not significantly contaminated with any of the other general factors. This is the first reported identification and purification of Drosophila TFIIH and TFIIE. Further analysis shows that, similar to their mammalian counterparts, Drosophila TFIIH is composed of eight polypeptides sized between 30 and 100 kDa, and Drosophila TFIIE is composed of two polypeptides sized at 34 and 60 kDa. When all of these fractions are combined with recombinant Drosophila TFlIB, a highly purified in vitro transcription system is generated that has not previously been available in Drosophila. The TFIID fraction can be replaced with recombinant Drosophila TBP to give a transcription system that is nearly free of contaminating proteins.

Colored diffraction catastrophes.

On fine scales, caustics produced with white light show vividly colored diffraction fringes. For caustics described by the elementary catastrophes of singularity theory, the colors are characteristic of the type of singularity. We study the diffraction colors of the fold and cusp catastrophes. The colors can be simulated computationally as the superposition of monochromatic patterns for different wavelengths. Far from the caustic, where the luminosity contrast is negligible, the fringe colors persist; an asymptotic theory explains why. Experiments with caustics produced by refraction through irregular bathroom-window glass show good agreement with theory. Colored fringes near the cusp reveal fine lines that are not present in any of the monochromatic components; these lines are explained in terms of partial decoherence between rays with widely differing path differences.

Lack of functional retinoblastoma protein mediates increased resistance to antimetabolites in human sarcoma cell lines.

Growth inhibition assays indicated that the IC50 values for methotrexate (MTX) and 5-fluorodeoxyuridine (FdUrd) in HS-18, a liposarcoma cell line lacking retinoblastoma protein (pRB), and SaOS-2, an osteosarcoma cell line with a truncated and nonfunctional pRB, were 10- to 12-fold and 4- to 11-fold higher, respectively, than for the HT-1080 (fibrosarcoma) cell line, which has wild-type pRB. These Rb-/- cell lines exhibited a 2- to 4-fold increase in both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) enzyme activities as well as a 3- to 4-fold increase in mRNA levels for these enzymes compared to the HT-1080 (Rb+/+) cells. This increase in expression was not due to amplification of the DHFR and TS genes. Growth inhibition by MTX and FdUrd was increased and DHFR and TS activities and expression were correspondingly decreased in Rb transfectants of SaOS-2 cells. In contrast, there was no significant difference in growth inhibition among these cell lines for the nonantimetabolites VP-16, cisplatin, and doxorubicin. A gel mobility-shift assay showed that parental SaOS-2 cells had increased levels of free E2F compared to the Rb-reconstituted SaOS-2 cells. These results indicate that pRB defective cells may have decreased sensitivity to growth inhibition by target enzymes encoded by genes whose transcription is enhanced by E2F proteins and suggest mechanisms of interaction between cytotoxic agents and genes involved in cell cycle progression.

The cis-acting phorbol ester "12-O-tetradecanoylphorbol 13-acetate"-responsive element is involved in shear stress-induced monocyte chemotactic protein 1 gene expression.

Vascular endothelial cells, serving as a barrier between vessel and blood, are exposed to shear stress in the body. Although endothelial responses to shear stress are important in physiological adaption to the hemodynamic environments, they can also contribute to pathological conditions--e.g., in atherosclerosis and reperfusion injury. We have previously shown that shear stress mediates a biphasic response of monocyte chemotactic protein 1 (MCP-1) gene expression in vascular endothelial cells and that the regulation is at the transcriptional level. These observations led us to functionally analyze the 550-bp promoter region of the MCP-1-encoding gene to define the cis element responding to shear stress. The shear stress/luciferase assay on the deletion constructs revealed that a 38-bp segment (-53 to -90 bp relative to the transcription initiation site) containing two divergent phorbol ester "12-O-tetradecanoylphorbol 13-acetate" (TPA)-responsive elements (TRE) is critical for shear inducibility. Site-specific mutations on these two sites further demonstrated that the proximal one (TGACTCC) but not the distal one (TCACTCA) was shear-responsive. Shear inducibility was lost after the mutation or deletion of the proximal site. This molecular mechanism of shear inducibility of the MCP-1 gene was functional in both the epithelial-like HeLa cells and bovine aortic endothelial cells (BAEC). In a construct with four copies of the TRE consensus sequences TGACTACA followed by the rat prolactin minimal promoter and luciferase gene, shear stress induced the reporter activities by 35-fold and 7-fold in HeLa cells and BAEC, respectively. The application of shear stress on BAEC also induced a rapid and transient phosphorylation of mitogen-activated protein kinases. Pretreatment of BAEC with TPA attenuated the shear-induced mitogen-activated protein kinase phosphorylation, suggesting that shear stress and TPA share a similar signal transduction pathway in activating cells. The present study provides a molecular basis for the transient induction of MCP-1 gene by shear stress.

The structure of Ascaris hemoglobin domain I at 2.2 A resolution: molecular features of oxygen avidity.

The perienteric hemoglobin of the parasitic nematode Ascaris has an exceptionally high affinity for oxygen. It is an octameric protein containing two similar heme-binding domains per subunit, but recombinant constructs expressing a single, monomeric heme-binding domain (domain 1; D1) retain full oxygen avidity. We have solved the crystal structure of D1 at 2.2 A resolution. Analysis of the structure reveals a characteristic globin fold and illuminates molecular features involved in oxygen avidity of Ascaris perienteric hemoglobin. A strong hydrogen bond between tyrosine at position 10 in the B helix (tyrosine-B10) and the distal oxygen of the ligand, combined with a weak hydrogen bond between glutamine-E7 and the proximal oxygen, grips the ligand in the binding pocket. A third hydrogen bond between these two amino acids appears to stabilize the structure. The B helix of D1 is displaced laterally by 2.5 A when compared with sperm whale myoglobin. This shifts the tyrosine-B10 hydroxyl far enough from liganded oxygen to form a strong hydrogen bond without steric hindrance. Changes in the F helix compared with myoglobin contribute to a tilted heme that may also be important for oxygen affinity.

Pancreatic digestive enzyme secretion in the rabbit: rapid cyclic variations in enzyme composition.

The role and mechanism of nonparallel pancreatic secretion of digestive enzymes, in which enzyme proportions change in rapidly regulated fashion, remain controversial. Secretion was collected from male 2.2-kg New Zealand rabbits in 5-min intervals for 3 h under basal conditions or constant stimulation with cholecystokinin (CCK; 0.1 microgram per kg per h i.v.) or methacholine chloride (MCh; 40 micrograms per kg per h i.v.). Both CCK and MCh produced an 8-fold stimulation of protein output. Enzymes were separated by SDS/PAGE and quantitated by densitometry of Coomassie blue-stained gels. Under both basal conditions and constant MCh infusion, rapid neurosecretory-like 12-min cyclic changes occurred in the proportions of amylase, lipase I, chymotrypsinogen, and trypsinogen. During constant infusion their percentages changed as much as 10-fold, and their ratios cycled by as much as 30-fold. The mean percentage for the entire infusion period for lipase I declined > 25% with CCK or MCh, for amylase it rose approximately 30%, and for chymotrypsinogen and trypsinogen it doubled (for all, P < 0.05). CCK and MCh elicited subtly but significantly different mean enzyme percentages and enzyme ratios (P < 0.05) for amylase, chymotrypsinogen, and trypsinogen; these differences were also confirmed by regression and correlation analyses. The changes in enzyme percentages and ratios were explicitly consistent with secretagogue-caused shifts in the intrapancreatic enzyme secretory sources. Nonparallel secretion of digestive enzymes occurs routinely, even during constant stimulation, and is due to cyclic neurosecretory-like secretion from heterogeneous intrapancreatic sources.

Nanomateriais luminescentes de terras raras utilizando complexos de benzenotricarboxilatos como precursores

O material Y2O3:Eu3+ vem sendo usado comercialmente como luminóforo vermelho desde da década de 1960, em uma grande variedade de aplicações devido ao seu elevado rendimento quântico (próximo de 100 %), elevada pureza de cor e boa estabilidade. Portanto, este trabalho propõe um novo método de síntese baseado nos complexos benzenotricarboxilatos (BTC) de terras raras trivalentes (RE3+) dopados com íons Eu3+. O objetivo principal é produzir materiais luminescente RE2O3:Eu3+ a temperatura mais baixa (500 °C) e em escala nanométrica. Os complexos precursores [RE(BTC):Eu3+] e [RE(TLA)·n(H2O):Eu3+], onde RE3+: Y, Gd e Lu; BTC: ácido trimésico (TMA) e ácido trimelítico (TLA) foram calcinados em diferentes temperaturas de 500 a 1000 °C, a fim de obter os materiais luminescentes RE2O3:Eu3+. Os complexos foram caracterizados por análise elementar de carbono e hidrogênio, analise térmica (TG), espectroscopia de absorção no infravermelho (FTIR), difração de raios-X - método do pó (XPD) e microscopia eletrônica de varredura (SEM). Todos os complexos são cristalinos e termo estáveis até 460 °C. Dados de fosforescência dos complexos de Y, Gd e Lu mostram que o nível T1 do aníon BTC3- tem energia acima do nível emissor 5D0 do íon Eu3+, indicando que os ligantes podem atuar como sensibilizadores de energia intramolecular. O estudo das propriedades fotoluminescentes dos complexos dopados foi baseado nos espectros de excitação e emissão e curvas de decaimento de luminescência. Ademais, foram determinados os parâmetros de intensidades experimentais (Ωλ), tempos de vida (τ), taxas de decaimentos radiativo (Arad) e não-radiativo (Anrad). Os materiais luminescentes RE2O3:Eu3+ foram sintetizados de forma bem sucedida por meio do método benzenotricarboxilatos calcinados a 500, 600, 700, 800, 900 e 1000 °C, apresentando alta homogeneidade química e controle de tamanho de cristalito. Os nanomateriais foram caracterizados pelas técnicas de FTIR, XPD SEM e TEM revelando a obtenção dos materiais C-RE2O3:Eu3+ mesmo a 500 °C. Os dados de XPD dos materiais confirmaram um aumento do tamanho dos cristalitos de 5 até 52 nm (equação de Scherrer) de em função da temperatura de calcinação de 500 a 1000 °C, respectivamente, corroborados pelas técnicas de SEM e TEM. Os espectros de emissão de RE2O3:Eu3+ mostram uma banda larga atribuída a transição interconfiguracional de transferência de carga ligante-metal (LMCT) em 260 nm, i.e. O2-(2p)→Eu3+(4f6). Além disso, foram observadas linhas finas de absorção devido as transições intraconfiguracionais 4f do íon európio (7F0,1𔾹LJ; J: 0, 1, 2, 3 e 4), como esperado. As propriedades fotoluminescentes dos luminóforos foram baseadas nos espectros (excitação e emissão) e curvas de decaimento luminescente. Os parâmetros de intensidade experimental, tempos de vida, assim como as taxas de decaimentos radiativos e não radiativos foram calculados. As propriedades fotônicas dos nanomateriais são consistentes com o sítio de baixa simetria C2 ocupado pelo íon Eu3+ no C-RE2O3:Eu3+, produzindo emissão vermelha dominada pela transição hipersensível 5D0𔾻F2 do íon Eu3+ no sitio C2, ao invés do sítio centrossimétrico S6. Além disso, os nanomateriais Y2O3:Eu3+ exibem características espectroscópicas semelhantes e elevados valores de eficiência quântica (η~91 %), compatível com os luminóforos comerciais disponíveis no mercado. Este novo método pode ser utilizado para o desenvolvimento de novos nanomateriais contendo íons terras raras, assim como outros íons metálicos.

Complexos de vanádio como miméticos de peroxidases e catalisadores de oxidação: síntese e caracterização espectroscópica

Quatro novos complexos mononucleares e um dinuclear de vanádio(IV) contendo ligantes do tipo imínico e carboxilato foram sintetizados e caracterizados através de diferentes técnicas espectroscópicas, UVNis, FT/IR e EPR, além de análise elementar, medidas de condutividade molar e, para alguns deles, análise termogravimétrica. Alguns deles foram obtidos como espécies neutras e outros, contendo grupos carboxilatos, foram isolados como espécies aniônicas, com contra-íons Na+ ou NH4+. Os complexos clássicos da literatura, largamente estudados e caracterizados, [VIVO(acac)2] e [VIvO(salen)], também foram sintetizados e caracterizados, a fim de comparar suas propriedades com aquelas dos novos complexos de vanádio(IV) sintetizados. Através das técnicas espectroscópicas, as principais bandas de transição e os principais grupos funcionais existentes nos complexos puderam ser verificados, bem como a simetria da estrutura geométrica e a confirmação do estado de oxidação do metal nos complexos. Além disso, através de medidas de condutividade molar e análise térmica foram confirmadas as razões estequiométricas ligante: metal em cada complexo, verificando-se, por exemplo, a natureza dimérica proposta para o complexo [VIVO(dbhab)] 2.

Análise da dinâmica e quantificação metabólica de imagens de medicina nuclear na modalidade PET/CT.

A presença da Medicina Nuclear como modalidade de obtenção de imagens médicas é um dos principais procedimentos utilizados hoje nos centros de saúde, tendo como grande vantagem a capacidade de analisar o comportamento metabólico do paciente, traduzindo-se em diagnósticos precoces. Entretanto, sabe-se que a quantificação em Medicina Nuclear é dificultada por diversos fatores, entre os quais estão a correção de atenuação, espalhamento, algoritmos de reconstrução e modelos assumidos. Neste contexto, o principal objetivo deste projeto foi melhorar a acurácia e a precisão na análise de imagens de PET/CT via processos realísticos e bem controlados. Para esse fim, foi proposta a elaboração de uma estrutura modular, a qual está composta por um conjunto de passos consecutivamente interligados começando com a simulação de phantoms antropomórficos 3D para posteriormente gerar as projeções realísticas PET/CT usando a plataforma GATE (com simulação de Monte Carlo), em seguida é aplicada uma etapa de reconstrução de imagens 3D, na sequência as imagens são filtradas (por meio do filtro de Anscombe/Wiener para a redução de ruído Poisson caraterístico deste tipo de imagens) e, segmentadas (baseados na teoria Fuzzy Connectedness). Uma vez definida a região de interesse (ROI) foram produzidas as Curvas de Atividade de Entrada e Resultante requeridas no processo de análise da dinâmica de compartimentos com o qual foi obtida a quantificação do metabolismo do órgão ou estrutura de estudo. Finalmente, de uma maneira semelhante imagens PET/CT reais fornecidas pelo Instituto do Coração (InCor) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP) foram analisadas. Portanto, concluiu-se que a etapa de filtragem tridimensional usando o filtro Anscombe/Wiener foi relevante e de alto impacto no processo de quantificação metabólica e em outras etapas importantes do projeto em geral.

Kinetic study of thermal 1,3-dipolar cycloaddition of azomethine ylides using differential scanning calorimetry as monitoring window

Kinetics of 1,3-dipolar cycloaddition involving azomethine ylides, generated from thermal [1,2]-prototropy of the corresponding imino ester, employing differential scanning calorimetry (DSC), is surveyed. Glycine and phenylalanine derived imino esters have different behavior. The first one prefers reacting with itself at 75 ºC, rather than with the dipolarophile. However, the α-substituted imino ester gives the cycloadduct at higher temperatures. The thermal dynamic analysis by 1H NMR of the neat reaction mixture of the glycine derivative reveals the presence of signals corresponding to the dipole in very small proportion. The non-isothermal and isothermal DSC curves of the cycloaddition of phenylalaninate and diisobutyl fumarate are obtained from freshly prepared samples. The application of known kinetic models and mathematical multiple non-linear regressions (NLR) allow to determine and to compare Ea, lnA, reaction orders, and reaction enthalpy. Finally a rate equation for each different temperature can be established for this particular thermal cycloaddition.

GU Monocerotis: A high-mass eclipsing overcontact binary in the young open cluster Dolidze 25

Context. The eclipsing binary GU Mon is located in the star-forming cluster Dolidze 25, which has the lowest metallicity measured in a Milky Way young cluster. Aims. GU Mon has been identified as a short-period eclipsing binary with two early B-type components. We set out to derive its orbital and stellar parameters. Methods. We present a comprehensive analysis, including B and V light curves and 11 high-resolution spectra, to verify the orbital period and determine parameters. We used the stellar atmosphere code FASTWIND to obtain stellar parameters and create templates for cross-correlation. We obtained a model to fit the light and radial-velocity curves using the Wilson-Devinney code iteratively and simultaneously. Results. The two components of GU Mon are identical stars of spectral type B1 V with the same mass and temperature. The light curves are typical of an EW-type binary. The spectroscopic and photometric analyses agree on a period of 0.896640 ± 0.000007 d. We determine a mass of 9.0 ± 0.6 M⊙ for each component and for temperatures of 28 000 ± 2000 K. Both values are consistent with the spectral type. The two stars are overfilling their respective Roche lobes, sharing a common envelope and, therefore the orbit is synchronised and circularised. Conclusions. The GU Mon system has a fill-out factor above 0.8, containing two dwarf B-type stars on the main sequence. The two stars are in a very advanced stage of interaction, with their extreme physical similarity likely due to the common envelope. The expected evolution of such a system very probably leads to a merger while still on the main sequence.

ejī:harī:nāme:harī:jāṇī:e : manuscript, 1829?

Title provided by cataloger.

Abordagem terapêutica da papilomatose laríngea : a propósito de um caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Tectonic evolution of the Proterozoic Itremo Group metasediments in central Madagascar

The major geologic units of the Itremo region in central Madagascar include: (1) upper amphibolite to granulite facies (higher grade) Precambrian rocks, mainly para- and orthogneisses, and migmatites; (2) the newly defined Itremo Nappes, a fold-and-thrust belt containing the Proterozoic Itremo Group sediments, metamorphosed at greenschist to lower amphibolite facies (lower grade) conditions: (3) Middle Neoproterozoic and Late Neoproterozoic-Cambrian intrusives. The stratigraphic succession of the Itremo Group in the eastern part of the Itremo region is, from bottom to top: quartzites, metapelites, metacarbonates and metapelites overlain by metacarbonates. During D1 the Itremo Group sediments were detached from their continental substratum, deformed into a fold-and-thrust nappe (Itremo Nappes), and transported on top of higher grade rocks that are intruded by Middle Neoproterozoic (c. 797–780 Ma) granites and gabbros. A second phase of deformation shortening (D2) affected both the Itremo Sedimentary Nappes and structurally underlying higher-grade rocksunits, and formed large-scale N-S-trending F2 folds. S1 axial plane foliations in Itremo Group sediments are truncated by Late Neoproterozoic-Cambrian granites (c. 570–540 Ma). The age of the formation of the Itremo Nappes is not well constrained: they formed in Neoproterozoic times between 780 and 570 Ma.