Generalization of spontaneous imitation from nonwords to real wordsa).

This study reports that English speakers, after shadowing English-like nonwords beginning on /p/ with extended voice onset time, spontaneously shifted their subsequent reading productions of English words converging toward the shadowing targets. The extent of the imitative changes correlated positively with the speakers' declarative memory of the nonwords, but not with the lexical frequency of the produced words. These findings provide evidence for the phoneme-level abstraction in perceptually induced phonetic drifts while they further suggest that the mechanisms underlying phonetic drifts in direct shadowing, and in subsequent productions of words differing from the shadowing targets, may not be identical.

Effect of shading and nitrogen fertilization on nitrogen metabolism, essential oil content and antimicrobial activity of Achillea millefolium

The Achillea millefolium L. is a perennial herb with important antibacterial, antifungal, anti-inflammatory, antitumoral, and antioxidant properties. This research aimed to investigate the effect of shading (75%; black net) and nitrogen fertilization (0, 75 and 150 kg urea ha-1) on the nitrogen metabolism, essential oil yield and antimicrobial activity of A.millefolium at vegetative- and reproductive-stage. The evaluated parameters varied depending on the organ and the phenological stage of the plant considered. Overall, our findings indicated that shading decreased nitrogen assimilation. Decreased activities of nitrate reductase and glutamine synthetase were observed on shaded plants during reproductive and vegetative stages, respectively. Nitrate and total amino acid levels increased in shaded plants at the vegetative stage. Regarding nitrogen supply, the improved nitrogen metabolism and essential oil yield values were accompanied by intermediate concentrations of urea (75 kg ha-1). Plants fertilized with 75 kg urea ha-1 produced the highest amino acids concentration (vegetative stage), ammonium concentration (vegetative stage) and essential oil yield (reproductive stage). Shading or nitrogen supply did not influence the microbial activity of A. millefolium essential oil.However, the essential oil of leaves and flowers were highly effective against fungi and bacteria, especially gram-positive bacteria. In conclusion, the current study showed that full light and 75 kg urea ha-1 enhanced the nitrogen metabolism of A. millefolium in both vegetative and reproductive stages.

Influence of light on the initial growth of invasive Cryptostegia madagascariensis Bojer in the Brazilian semiarid region / Influência da luz no crescimento inicial da invasora Cryptostegia madagascariensis Bojer no semiárido Brasileiro

The invasion by Cryptostegia madagascariensis causes economic and environmental problems in Northeastern Brazil. Current study evaluates the initial growth of C. madagascariensis on light gradient. The experiment used 0, 30, 50 and 70% shading treatments arranged in a completely randomized design and evaluated in four periods, namely, 35, 50, 65 and 80 days. Height, absolute growth rate, relative growth rate, number of side branches, number of leaves, leaf area, specific leaf area, leaf area ratio, root mass ratio, stem mass ratio, leaf mass ratio and leaf area/root mass ratio were evaluated. Results showed that the height was greater in shade environments, and the greater the available light, the greater was the number of side branches. C. madagascariensis is able to change the leaf area, specific leaf area and biomass allocation in different shadings. Since C. madagascariensis varies growth forms and performs morphological adjustments to increase light uptake, the plant has warranted success during the initial growth under different shadings.

A invasão da Cryptostegia madagascariensis causa problemas econômicos e ambientais no Nordeste do Brasil. O objetivo do trabalho foi estudar o crescimento inicial da C. madagascariensis em gradiente de luz. O experimento utilizou os tratamentos de 0, 30, 50 e 70% de sombreamento, dispostos em um delineamento experimental inteiramente casualizado e avaliados em quatro períodos: 35, 50, 65 e 80 dias. Foram avaliados a altura, a taxa de crescimento absoluto, a taxa de crescimento relativo, o número de ramos laterais e de folhas, a área foliar, a área foliar específica, a razão de área foliar, a razão de massa da raiz, a razão de massa do caule, a razão de massa das folhas e a razão área foliar/massa da raiz. Os resultados mostraram que a altura foi maior em ambientes sombreados e, quanto mais luz disponível, maior o número de ramos laterais. A C. madagascariensis é capaz de alterar a área foliar, a área foliar específica e na alocação de biomassa nos diferentes sombreamentos. Portanto, a C. madagascariensis varia a forma de crescimento e realiza ajustes morfológicos para aumentar a captação de luz, o que garantiu seu sucesso durante o crescimento inicial em diferentes sombreamentos.

Lâminas de irrigação e sombreamento na produção de biomassa de Acariçoba (Hydrocotyle umbellata L. ) / Levels of irrigation and shade in the production of the biomass of dollarweed (Hydrocotyle umbellata L. )

RESUMO: A acariçoba é uma planta utilizada pela medicina ayurvédica e em cosméticos por possuir propriedades rejuvenescedoras e revitalizante do sistema nervoso e cérebro. Com objetivo de avaliar os caracteres agronômicos e o teor de flavonóides totais de acariçoba em resposta a aplicação de lâminas distintas de irrigação e sombreamento, foi conduzido um experimento em área localizada nas coordenadas 17º 47' 53'' latitude (S); longitude (W) - 51º 55' 53'', com altitude média de 648m, no período de julho a novembro. O clima é Aw (tropical), relevo suave ondulado (8% de declividade) com solo classificado como Latossolo Vermelho Distroférrico e textura argilosa. As temperaturas médias anuais são 18,1ºC e 30ºC. A umidade relativa do ar média é de 64,7% e a precipitação total anual média é de 1550 mm, com estações do ano seca e chuvosa, bem definidas. O ensaio foi conduzido em blocos casualizados em esquema fatorial 2 x 2, com 5 repetições totalizando vinte unidades experimentais com dois manejos de irrigação, sendo um com aplicação de uma lâmina de água diariamente, e outro, com duas aplicações de lâmina de água diariamente. Os tratamentos de sombreamento foram a pleno sol e com 50% de sombreamento. Foram avaliadas a massa fresca e massa seca das partes aéreas, a altura e o número de plantas, e o teor de flavonóides totais das partes aéreas. A produção de flavonóides foi favorecida pelo sombreamento, enquanto a irrigação não influenciou nem na produção de flavonoides, nem nos caracteres agronômicos avaliados.

ABSTRACT: Dollarweed is a plant that is widely used by the Ayurvedic medicine and in the cosmetics industry because of its rejuvenating and revitalizing properties related to the nervous system and the brain. In order to evaluate the agronomic characteristics and the content of total flavonoids of the dollarweed, in response to the application of different levels of irrigation and shade, an experiment was conducted in an area at the coordinates 17º 47' 53'' latitude (S), 51º 55' 53'' longitude (W), with a mean altitude of 648m, from July to November. The climate is Aw (tropical), gently undulating relief (8% slope) with distroferric red latosol and clayey texture. The average annual temperatures are 18.1 ºC and 30 ºC. The average relative humidity of the air is 64.7% and the average annual rainfall is 1550 mm, with well-defined dry and rainy seasons. The assay was in randomized blocks, with a 2 x 2 factorial design, with 5 repetitions, amounting to twenty experimental units with two irrigation managements, being one with a daily application of water and the other with two daily applications of water. The shading treatments were under full sun and 50% shade. The fresh and dry weights of the aerial parts, the height and number of plants and the total content of flavonoids of the aerial parts were evaluated. The production of flavonoids was favored by the shade; irrigation did not influence the production of flavonoids or the agronomic characters evaluated.

Efeitos do sombreamento na anatomia foliar de Gallesia integrifolia (Spreng) Harms e Schinnus terebinthifolius Raddi / Effects of shading on leaf anatomy of Gallesia integrifolia (Spreng) Harms and Schinnus terebinthifolius Raddi

Realizou-se estudo com o objetivo de analisar as alterações na anatomia foliar de Gallesia integrifolia (Spreng) Harms e Schinnus terebinthifolius Raddi quando cultivadas em ambientes de sombra moderada ou densa, simulando as condições naturais encontradas em sistemas agroflorestais tradicionais do sul da Bahia, Brasil. Plantas das duas espécies, com aproximadamente um ano de idade, foram cultivadas em casa de vegetação sob quatro níveis de sombreamento (25%, 17%, 10% e 5%). Estudos anatômicos do limbo foliar foram realizados a partir de material incluído em parafina e seccionado em micrótomo rotativo. Os diferentes níveis de sombreamento ocasionaram alterações na estrutura do mesofilo de ambas as espécies, com diferenças significativas na espessura do parênquima paliçádico, limbo foliar, e densidade estomática. Nas condições em que o experimento foi realizado os resultados obtidos indicaram que G. integrifolia apresenta maior capacidade de aclimatação a ambientes de sombra moderada e densa do que S. terebinthifolius, sendo mais indicada para o cultivo em sistemas agroflorestais pré-estabelecidos.

This study was conducted with the aim of analyzing the changes in the leaf anatomy of Gallesia integrifolia (Spreng) Harms and Schinnus terebinthifolius Raddi when grown in environments with moderate to dense shade, simulating the natural conditions found in traditional agroforestry systems in southern Bahia, Brazil. Plants of both species, being approximately one year-old, were grown at four irradiance levels (25%, 17%, 10% and 5%) in a greenhouse. Leaf anatomical studies were made from material embedded in paraffin and sectioned on a rotary microtome. The different irradiance levels caused a shift in the mesophyll anatomy of both species, with significant differences in palisade parenchyma thickness and leaf stomatal density. In the conditions of this experiment, the results indicated that G. integrifolia has a higher capacity for acclimatization to moderate to dense shade than S. terebinthifolius, and it is thus more suitable for cultivation in pre-established agroforestry systems.

Desenvolvimento de Maytenus ilicifolia e de seus polifenóis totais sob condição de sombreamento e poda / Shadowing and pruning on the development and polyphenol contents in Maytenus ilicifolia

Maytenus ilicifolia Mart. ex Reissek (Celastraceae), conhecida popularmente por espinheira-santa, é nativa e cultivada, principalmente, no sul do Brasil. Suas folhas apresentam triterpenos e substâncias polifenólicas (flavonóides e taninos) relacionadas ao efeito antiulcerogênico. O objetivo do trabalho foi avaliar a produção de massa seca, altura, ramificação, e o teor de polifenóis totais em plantas de M. ilicifolia que se desenvolveram sob duas condições de luminosidade, após dois tratamentos distintos de poda. O delineamento experimental foi em blocos cazualizados com plantas desenvolvidas sob sombra e a pleno sol, e com plantas manejadas por poda parcial e rasa, com quatro repetições. No tratamento de poda rasa a maior altura das plantas foi observada nas plantas cultivadas a sombra em comparação com as conduzidas a pleno sol. No tratamento de poda parcial o número de ramos terciários aumentou significativamente nas plantas conduzidas a pleno sol. As plantas cultivadas a pleno sol apresentaram diferença significativa na concentração de polifenóis totais em relação às plantas produzidas à sombra alcançando valores de 10,29 ± 0,20% (CV=1,94%) e 7,16 ± 0,09% (CV=1,30%) respectivamente.

The Maytenus ilicifolia Mart. ex Reissek (Celastraceae), traditionally known as "espinheira-santa" in Portuguese, is native and cultivated in South Brazil. Its leaves contain triterpenes and polyphenolic compounds (flavonoids and tannins), which are related to the antiulcerogenic effect. The aim of this work was to evaluate the dried biomass production (g), height (m), ramification, and also the total content in polyphenol compounds of the leaves from the M. ilicifolia specie, which were developed under two different light conditions after two different pruning treatments. The design of the experiment was randomized blocks with two levels of light (shadow or full sunlight) and two levels of prune (partial and drastic), with four replications. The height of the plants grown in the shadow was greater for the treatment of drastic pruning than in plants in full sunlight. The ramification increased significantly with drastic prune in full sunlight. The plants grown in full sunlight showed a significant higher content in total polyphenols than the plants grown in the shadow, 10.29±0.20% (RSD=1.94%) and 7.16±0.09% (RSD=1.30%) respectively.

Bilateral miliary shadowing on chest X-ray.

A patient presented with recent onset of increasing shortness of breath, weight loss and low-grade fever. His chest X-ray revealed bilateral miliary shadowing. He was investigated with CT-scanning of thorax. Later, a biopsy from supra-clavicular node and its immunocytochemistry studies confirmed metastasis from primary lung cancer. Primary lung cancer with miliary pulmonary metastases is a rare happening and is mostly associated with lung adenocarcinoma.

The effect of firearm muzzle gases on the backspatter of blood.

Injuries caused by gunshots can produce what bloodstain pattern analysts know as "backspatter." Observations about the presence or absence of backspatter on an individual may be used in court as evidence of guilt or innocence. The discharge of three firearms (.22 caliber revolver, .38 caliber revolver, and .308 caliber rifle) and the resulting impact of bullets on a blood source were recorded using high-speed digital video imaging. Blood droplets, firearm muzzle gases, and ballistic shock waves were visualized using standard reflected light and shadowgraphy imaging techniques. A significant interaction between air currents, muzzle gases, and particulate material emanating from the firearms upon discharge with backspattered blood was observed. Blood droplets, initially spattered back toward the firearm and the shooter, were observed to change direction under the influence of firearm-induced air currents and were blown forward toward and beyond their original source location. Implications for experts testifying in court and for bloodstain pattern instructors are discussed.

Nano-structure of the laminin γ-1 short arm reveals an extended and curved multidomain assembly.

Laminins are multidomain glycoproteins that play important roles in development and maintenance of the extracellular matrix via their numerous interactions with other proteins. Several receptors for the laminin short arms revealed their importance in network formation and intercellular signaling. However, both the detailed structure of the laminin γ-1 short arm and its organization within the complexes is poorly understood due to the complexity of the molecule and the lack of a high-resolution structure. The presented data provide the first subatomic resolution structure for the laminin γ-1 short arm in solution. This was achieved using an integrated approach that combined a number of complementary biophysical techniques such as small angle X-ray scattering (SAXS), analytical ultracentrifugation, dynamic light scattering and electron microscopy. As a result of this study, we have obtained a significantly improved model for the laminin γ-1 short arm that represents a major step forward in molecular understanding of laminin-mediated complex formations.

Desenvolvimento inicial de plântulas de Theobroma grandiflorum (Willd. ex Spreng.) Schum. sob influência de sombreamento / Initial development of Theobroma grandiflorum (Willd. ex Spreng.) Schum. saplings under shading influence

O presente estudo foi conduzido com o objetivo de avaliar o comportamento inicial de plântulas de Theobroma grandiflorum (Willd. ex Spreng.) Schum. (cupuaçu), em função de diferentes níveis de sombreamento. Ao final de 50 dias, após a emergência, as plântulas de cupuaçu foram submetidas a três níveis de sombreamento, sendo: 0 por cento de sombreamento, 50 por cento de sombreamento e Sombra Natural. O crescimento das mudas foi conduzido em delineamento de blocos casualizados, com 15 repetições, sendo cada planta considerada como uma repetição. Foram avaliados a altura, o diâmetro e o número de folhas aos 60, 82, 103, 124, 145 dias após a emergência das plântulas. A Massa Seca de Folhas (MSF), Massa Seca do Caule (MSC), Massa Seca da Raiz (MSR) e Massa Seca Total (MST), Relação parte aérea/raiz (PA/R) e relação Altura da planta/Diâmetro do colo (A/D), foram avaliadas no final do experimento. O crescimento inicial de Theobroma grandiflorum foi corroborado com os padrões da espécie, que ocorre no interior das matas primárias, tendo melhor desenvolvimento dos parâmetros avaliados em condições de 50 por cento de sombreamento. A condição de 50 por cento de sombreamento pode ser recomendada para a formação de mudas de Theobroma grandiflorum, devido o seu melhor desempenho em altura, diâmetro, número de folhas e alocação de massa nas partes da planta.

The objective of this work was to evaluate the initial growth of sapling of Theobroma grandiflorum (cupuaçú), in function of different levels of solar radiation. Fifty days, after the emergency, the saplings were submitted to three levels of shade, being: 0 percent, 50 percent and natural shade. Sapling development was carried out in randomized block design with 15 repetitions (15 saplings). The sapling height, lap diameter and the numbers of leaves being evaluated on the 60st, 82nd, 103rd, 124th, 145 days after sapling emergence. The Dry Mass of the leaves (DML), Dry Mass of the stem (DMS), Dry Mass of the root (DMR) and Dry mass of the plant (DMP), Relation shoot/root (PA/R) and relation plant height/Lap diameter, were measured at the end of the experiment, when the sapling reached the appropriate size to be planted in the field. The initial growth of Theobroma grandiflorum was corroborated with the species standard that occurs inside of the primary bushes, so its development of the parameters evaluated is better in conditions of 50 percent of shade. The condition of 50 percent of shade can be recommended for the formation of plants of Theobroma grandiflorum, which had the better performance in plant height, lap diameter, number and allocation of mass in the parts of the plant.

The prodomain of BMP-7 targets the BMP-7 complex to the extracellular matrix.

Biochemical and biophysical methods are used to show that BMP-7 is secreted as a stable complex consisting of the processed growth factor dimer noncovalently associated with its two prodomain propeptide chains and that the BMP-7 complex is structurally similar to the small transforming growth factor beta (TGFbeta) complex. Because the prodomain of TGFbeta interacts with latent TGFbeta-binding proteins, a family of molecules homologous to the fibrillins, the prodomain of BMP-7 was tested for binding to fibrillin-1 or to LTBP-1. The BMP-7 prodomain and BMP-7 complex, but not the separated growth factor dimer, interact with N-terminal regions of fibrillin-1. This interaction may target the BMP-7 complex to fibrillin microfibrils in the extracellular matrix. Immunolocalization of BMP-7 in tissues like the kidney capsule and skin reveals co-localization with fibrillin. However, BMP-7 immunolocalization in other tissues known to be active sites for BMP-7 signaling is not apparent, suggesting that immunolocalization of BMP-7 in certain tissues represents specific extracellular storage sites. These studies suggest that the prodomains of TGFbeta-like growth factors are important for positioning and concentrating growth factors in the extracellular matrix. In addition, they raise the possibility that prodomains of other TGFbeta-like growth factors interact with fibrillins and/or LTBPs and are also targeted to the extracellular matrix.

High resolution shadowing of Mycobacterium leprae.

Metal shadow casting techniques for transmission electron microscopic examination was used to determine the morphological characteristics of Mycobacterium leprae in untreated and treated patients. This technique is used to visualize bacterial surface structures by thermal evaporation of platinum alloys under moderate vacuum. This method gives a high contrast image at relatively low resolution and is useful for correlating micro-morphology quantitatively to early therapeutic effects of anti-leprosy drugs. Using these techniques in untreated cases, the surface structures of M. leprae were uniformly filled with relatively homogenous protoplasm surrounded by a cell wall. Most of the bacilli had thick cell walls with prominent banded and fibrous structures on the surface of the cell body. The cell wall was not detached in any of the solid bacilli in untreated cases. The bacilli varied in size and some of them were swollen in their mid-portion. Some bacilli were very short and completely filled with cytoplasm; therefore, these short bacilli were counted as solid bacilli in electron microscopic morphological index (EM-MI) determination. During treatment, mainly the cytoplasms of the bacilli were affected, and degeneration was observed. Ultrastructurally, the cytoplasm was shrunken and detached from the cell wall indicating mild degeneration. After moderate degeneration, the cytoplasm appeared fragmented. In advanced degeneration, all structures except the cell walls collapsed completely and no fibrous or band structures were visible on the surfaces of the cell walls. Therefore, these bacilli were counted as non-solid bacilli for EM-MI determination. This study shows that transmission electron shadowing gives more accurate counts than standard light microscopy of intact M. leprae bacilli in patient specimens.

Cuticular Architecture of Hassalstrongylus epsilon (Nematoda: Trichostrongyloidea).

Hassalstrongylus epsilon is a small nematode, whose adult forms are found among the intestinal microvilli of the water rat Nectomys squamipes, Brants 1827 (Rodentia: Muridae). The external appearance of the cuticle, which presents transversal striations and longitudinal ridges, is described using scanning electron microscopy. Transmission electron microscopy of thin sections and replicas of quick-frozen, freeze-fractured, deep-etched and rotatory shadowed samples showed the presence in the cuticle of struts that arise from the fluid median layer, extending outward to the epicuticle. The cuticle showed the presence of five layers: epicuticle, cortical, fibril-rich, fluid median and fibrous. The cuticle layers were made of an assemblage of fibers that create compartments, which were larger in the fluid region than in the fibril-rich median layer.

Crosslinking of fibrinogen and fibronectin by free radicals: a possible initial step in adhesion formation in osteoarthritis of the temporomandibular joint.

PURPOSE: Adhesion formation in osteoarthritis (OA) of the temporomandibular joint (TMJ) typically results in a sustained limitation of joint movement. We propose the hypothesis that free-radical-mediated crosslinking of proteins underlies this adhesion formation in affected joints. Free radicals may cause oxidative modification of proteins, creating an opportunity for the formation of intramolecular and intermolecular crosslinks via covalent bonds. This may stabilize protein aggregates, rendering them more resistant to degradation. In this study, the free-radical-mediated crosslinking of model proteins (fibrinogen and fibronectin) was investigated to test our hypothesis that free radicals contribute to adhesion formation via this mechanism in OA of the TMJ. MATERIALS AND METHODS: Physiological clot formation of fibrinogen by thrombin and free-radical-induced crosslinking of fibrinogen and of fibronectin were analyzed using spectrophotometric turbidity measurements, light-scattering techniques, polyacrylamide gel electrophoresis (PAGE), and rotary shadowing. RESULTS: Fibrinogen was shown to aggregate after free radical treatment, as detected using turbidity measurements and light-scattering techniques. Using PAGE, fibrinogen as well as fibronectin was shown to degrade under low oxidative stress. Under high oxidative stress, however, fragments from both proteins were found to be covalently crosslinked, resulting in high-molecular-weight protein aggregates. The aggregation was shown to be at random with rotary shadowing. CONCLUSION: The study shows that high oxidative stress contributes to the formation of crosslinked proteins that may serve as an initial scaffolding for the development of adhesions frequently seen in OA of the TMJ.

Structure of functional single AQP0 channels in phospholipid membranes.

Aquaporin-0 (AQP0) is the most prevalent intrinsic protein in the plasma membrane of lens fiber cells where it functions as a water selective channel and also participates in fiber-fiber adhesion. We report the 3D envelope of purified AQP0 reconstituted with random orientation in phospholipid bilayers as single particles. The envelope was obtained by combining freeze-fracture, shadowing and random conical tilt electron microscopy followed by single particle image processing. Two-dimensional analysis of 2547 untilted images produced eight class averages exhibiting "square" and "octagonal" shapes with a continuum of variation. We reconstructed in 3D five class averages that best described the data set. The reconstructions ("molds") appeared as metal cups exhibiting external and internal surfaces. We used the internal surface of the mold to calculate the "imprints" that represent the AQP0 particles protruding from the hydrophobic core of the phospholipid bilayer. The complete envelope of the channel, formed by joining the square and octagonal imprints, described accurately the size, shape, oligomeric state, orientation, and molecular weight of the AQP0 channel inserted in the phospholipid bilayer. Rigid body docking of the atomic model of the aquaporin-1 (AQP1) tetramer showed that the freeze-fracture envelope accounted for the conserved transmembrane domain (approximately 73% similarity between AQP0 and AQP1) but not for the amino and carboxyl termini. We suggest that the discrepancy might reflect differences in the location of the amino and carboxyl termini in the crystal and in the phospholipid bilayer.

Utrophin binds laterally along actin filaments and can couple costameric actin with sarcolemma when overexpressed in dystrophin-deficient muscle.

Dystrophin is widely thought to mechanically link the cortical cytoskeleton with the muscle sarcolemma. Although the dystrophin homolog utrophin can functionally compensate for dystrophin in mice, recent studies question whether utrophin can bind laterally along actin filaments and anchor filaments to the sarcolemma. Herein, we have expressed full-length recombinant utrophin and show that the purified protein is fully soluble with a native molecular weight and molecular dimensions indicative of monomers. We demonstrate that like dystrophin, utrophin can form an extensive lateral association with actin filaments and protect actin filaments from depolymerization in vitro. However, utrophin binds laterally along actin filaments through contribution of acidic spectrin-like repeats rather than the cluster of basic repeats used by dystrophin. We also show that the defective linkage between costameric actin filaments and the sarcolemma in dystrophin-deficient mdx muscle is rescued by overexpression of utrophin. Our results demonstrate that utrophin and dystrophin are functionally interchangeable actin binding proteins, but that the molecular epitopes important for filament binding differ between the two proteins. More generally, our results raise the possibility that spectrin-like repeats may enable some members of the plakin family of cytolinkers to laterally bind and stabilize actin filaments.

Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins.

Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500-A-long secreted protein that is rich in beta-structure and contains two regions, R1 and R2, of tandem 19-residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel beta-sheets; or (ii) a beta-helix in which the polypeptide chain is coiled to form three long parallel beta-sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional "covert" copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the beta-helix model that the shaft domain--a 350 A rod--should consist of a continuous run of these motifs, apart from loop inserts. The N-terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin-like structure. Drawing on crystal structures of known beta-helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins--all large secreted proteins of bacterial provenance--that have similar repeats and probably also similar structures.

XMAP215 is a long thin molecule that does not increase microtubule stiffness.

XMAP215 is a microtubule associated protein that speeds microtubule plus end growth by seven- to tenfold and protects these ends from destabilization by the Kin I kinesin, XKCM1. To understand the mechanisms responsible for these activities, it is necessary to know the structure of XMAP215. By unidirectional shadowing and electron microscopy, XMAP215 appeared as an elongate molecule of 60+/-18 nm, suggesting that XMAP215 could span up to seven to eight tubulin dimers along a protofilament. Most XMAP215 molecules were straight but a subset were bent suggesting that XMAP215 is flexible. Antibodies to the C terminus labeled one end of XMAP215 with no evidence for XMAP215 dimerization. Incubation of XMAP215 and tubulin at 4 degrees C resulted in assembly of curved protofilaments, which appeared to be incomplete tubulin rings. Measurements from rotary shadowed samples showed that tubulin/XMAP215 partial rings had an average width of 8.8+/-1.8 nm compared with 5.6+/-1.1 nm for rings assembled from tubulin dimers alone, suggesting that XMAP215 adds a width of approximately 3.2 nm to the curved tubulin protofilament. XMAP215 did not change the radius of curvature of these partial tubulin rings. Measurements of microtubule flexural rigidity by thermal fluctuations showed that XMAP215 did not change microtubule rigidity. Finally, sequence analysis shows that the N-terminal half of XMAP215 contains four repeats, each composed of multiple HEAT repeats.

Structural conversion between open and closed forms of radixin: low-angle shadowing electron microscopy.

The function of ERM (ezrin/radixin/moesin) proteins as general cross-linkers between actin filaments and plasma membranes is regulated downstream of Rho, through the transition between active and inactive forms. To directly examine the conformational change between the active and inactive forms of ERM proteins, we applied low-angle rotary-shadowing electron microscopy to the radixin molecules, wild-type, T564A-non-phosphorylated-type, and T564E-phosphorylated-type, since most of the active forms are reportedly stabilized in cells by the C-terminal threonine phosphorylation. As a result, the T564A- and wild-type radixin molecules yielded the globular closed forms, approximately 8-14 nm in diameter, with some striations on their surfaces. In contrast, the T564E-radixin molecules tended to take elongated open forms, in which two globular structures measuring approximately 8 nm and approximately 5 nm in diameter were associated with both ends of the filamentous structures. The filamentous structure took either a approximately 20-25 nm-long straight course or a folded course. Taken together with the biochemical and the crystal structural results obtained to date, the closed and open forms represent the inactive and active forms of radixin as cross-linkers between actin filaments and plasma membranes.

Electron microscopic observation and single-stranded DNA binding activity of the Mcm4,6,7 complex.

Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase activity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex was visualized by electron microscopy after negative staining with uranyl acetate. The complex contained toroidal forms with a central channel and also contained structures with a slit. Gel-shift analysis indicated that the level of affinity of the Mcm4,6,7 complex for single-stranded DNA was comparable to that of SV40 T antigen, although the Mcm4,6,7 complex required longer single-stranded DNA for the binding than did SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-stranded DNA were visualized as beads in a queue or beads on string-like structures. The formation of these nucleoprotein complexes was erased by Mcm2 that is a potential inhibitor of the Mcm4,6,7 helicase. We also found that the DNA helicase activity of Mcm4,6,7 complex was inhibited by the binding of Mcm3,5 complex. These results support the notion that the Mcm4,6,7 complex functions as a DNA helicase and the formation of 600 kDa complex is essential for the activity.