Actin-Bundling Protein Isolated from Pollen Tubes of Lily : Biochemical and Immunocytochemical Characterization

A 135-kD actin-bundling protein was purified from pollen tubes of lily (Lilium longiflorum) using its affinity to F-actin. From a crude extract of the pollen tubes, this protein was coprecipitated with exogenously added F-actin and then dissociated from F-actin by treating it with high-ionic-strength solution. The protein was further purified sequentially by chromatography on a hydroxylapatite column, a gel-filtration column, and a diethylaminoethyl-cellulose ion-exchange column. In the present study, this protein is tentatively referred to as P-135-ABP (Plant 135-kD Actin-Bundling Protein). By the elution position from a gel-filtration column, we estimated the native molecular mass of purified P-135-ABP to be 260 kD, indicating that it existed in a dimeric form under physiological conditions. This protein bound to and bundled F-actin prepared from chicken breast muscle in a Ca2+-independent manner. The binding of 135-P-ABP to actin was saturated at an approximate stoichiometry of 26 actin monomers to 1 dimer of P-135-ABP. By transmission electron microscopy of thin sections, we observed cross-bridges between F-actins with a longitudinal periodicity of 31 nm. Immunofluorescence microscopy using rhodamine-phalloidin and antibodies against the 135-kD polypeptide showed that P-135-ABP was colocalized with bundles of actin filaments in lily pollen tubes, leading us to conclude that it is the factor responsible for bundling the filaments.

Three quaternary structures for a single protein.

The structure of a multisubunit protein (immunoglobulin light chain) was solved in three crystal forms, differing only in the solvent of crystallization. The three structures were obtained at high ionic strength and low pH, high ionic strength and high pH, and low ionic strength and neutral pH. The three resulting "snapshots" of possible structures show that their variable-domain interactions differ, reflecting their stabilities under specific solvent conditions. In the three crystal forms, the variable domains had different rotational and translational relationships, whereas no alteration of the constant domains was found. The critical residues involved in the observed effect of the solvent are tryptophans and histidines located between the two variable domains in the dimeric structure. Tryptophan residues are commonly found in interfaces between proteins and their subunits, and histidines have been implicated in pH-dependent conformation changes. The quaternary structure observed for a multisubunit protein or protein complex in a crystal may be influenced by the interactions of the constituents within the molecule or complex and/or by crystal packing interactions. The comparison of buried surface areas and hydrogen bonds between the domains forming the molecule and between the molecules forming the crystals suggest that, for this system, the interactions within the molecule are most likely the determining factors.

Human plectin: organization of the gene, sequence analysis, and chromosome localization (8q24).

Plectin, a 500-kDa intermediate filament binding protein, has been proposed to provide mechanical strength to cells and tissues by acting as a cross-linking element of the cytoskeleton. To set the basis for future studies on gene regulation, tissue-specific expression, and pathological conditions involving this protein, we have cloned the human plectin gene, determined its coding sequence, and established its genomic organization. The coding sequence contains 32 exons that extend over 32 kb of the human genome. Most of the introns reside within a region encoding the globular N-terminal domain of the molecule, whereas the entire central rod domain and the entire C-terminal globular domain were found to be encoded by single exons of remarkable length, >3 kb and >6 kb, respectively. Overall, the organization of the human plectin gene was strikingly similar to that of human bullous pemphigoid antigen 1 (BPAG1), confirming that both proteins belong to the same gene family. Comparison of the deduced protein sequences for human and rat plectin revealed that they were 93% identical. By using fluorescence in situ hybridization, we have mapped the plectin gene to the long arm of chromosome 8 within the telomeric region. This gene locus (8q24) has previously been implicated in the human blistering skin disease epidermolysis bullosa simplex Ogna. Detailed knowledge of the structure of the plectin gene and its chromosome localization will aid in the elucidation of whether this or any other pathological conditions are linked to alterations in the plectin gene.

Functional protection of dystrophic mouse (mdx) muscles after adenovirus-mediated transfer of a dystrophin minigene.

Fast skeletal muscles of mdx (X chromosome-linked muscular dystrophy) mice were injected after birth with a recombinant adenovirus containing a minidys- trophin gene, a 6.3-kbp cDNA coding for the N- and C-terminal ends of dystrophin. Adult muscles were challenged by forced lengthening during tetanic contractions. Stretch-induced mechanical and histological damages were much reduced in injected muscles, in direct proportion of the Miniber of fibers expressing minidystrophin. Damaged fibers were preferentially found among minidystrophin-negative regions. Minidystrostrophin confers an important functional and structural protection of limb muscles against high mechanical stress, even after a partial somatic gene transfer.

Mammalian phospholipase D: activation by ammonium sulfate and nucleotides.

Phospholipase D (PLD) associated with the rat kidney membrane was activated by guanine 5'-[gamma-thio]triphosphate and a cytosol fraction that contained ADP-ribosylation factor. When assayed by measuring the phosphatidyl transfer reaction to ethanol with exogenously added radioactive phosphatidylcholine as substrate, the PLD required a high concentration (1.6 M) of ammonium sulfate to exhibit high enzymatic activity. Other salts examined were far less effective or practically inactive, and this dramatic action of ammonium sulfate is not simply due to such high ionic strength. Addition of ATP but not of nonhydrolyzable ATP analogue adenosine 5'-[beta, gamma-imido]diphosphate further enhanced the PLD activation approximately equal to 2- to 3-fold. This enhancement by ATP needed cytosol, implying a role of protein phosphorylation. A survey of PLD activity in rat tissues revealed that, unlike in previous observations reported thus far, PLD was most abundant in membrane fractions of kidney, spleen, and liver in this order, and the enzymatic activity in brain and lung was low.

Association of a transglutaminase-related antigen with intermediate filaments.

A mouse monoclonal antibody, G92.1.2, raised against guinea pig liver transglutaminase (TGase) recognizes an antigen present in primary mouse dermal fibroblasts. A filamentous pattern, bearing remarkable similarity to the vimentin intermediate filament (IF) network, is seen when these cells are fixed and processed for indirect immunofluorescence with the antibody. Double-label immunofluorescence reveals that the antigen reacting with the antibody colocalizes precisely with vimentin IF and that this colocalization is retained after the treatment of fibroblasts with colchicine, which induces a redistribution of the majority of IFs into perinuclear aggregates. These morphological observations are further supported by the finding that the protein reacting with G92.1.2 is retained in IF-enriched cytoskeletal preparations made by using nonionic detergent-containing high ionic strength solutions. Western blots of the IF fraction show that G92.1.2 recognizes a major band of approximately 280 kDa and does not cross react with vimentin. Furthermore, when the antibody is microinjected into live dermal fibroblasts, it causes a collapse of the vimentin IF network in the majority of injected cells. The results suggest that a form of TGase, or a TGase-related antigen, is closely associated with the vimentin IF network of primary cultures of mouse dermal fibroblasts.

Aplicação de gelatina obtida de subproduto animal como substituto parcial de gordura em spread de chocolate

O objetivo do presente estudo foi verificação da viabilidade da aplicação de gelatina de pés de frango em spread de chocolate como substituto parcial de gordura. Inicialmente apresentou-se uma revisão de literatura sobre os principais aspectos que envolvem a pesquisa. Após relatou-se a metodologia de extração e caracterização físico-química da gelatina obtida de pés de frango por meio de análises de composição centesimal, de cor, força de gel e análise de perfil de textura (TPA). Posteriormente abordou-se os efeitos da aplicação de gelatina como substituto de gordura nas propriedades físico-químicas e reológicas de spread de chocolate. Por último investigou-se a influência da concentração de gelatina e substituição de gordura na espalhabilidade, estabilidade (shelf life), custos e propriedades sensoriais dos spreads de chocolate. Para tanto, foi utilizado planejamento experimental Central Composto Rotacional e metodologia de superfície de resposta. Os resultados da extração de gelatina de subproduto de frango indicaram que as peles e tendões dos pés de frango possuem rendimento (7,83 %) e conteúdo mineral (1,92 %), apresentando alto teor proteico (84,96 %) próximo das gelatinas comerciais. Em concentração de 6,67 % a gelatina demonstrou elevada força de gel (294,78 g), comportando-se na análise de perfil de textura como um sólido. Os dados relacionados às formulações de spread de chocolate demonstraram que tanto o nível de substituição de gordura quanto a concentração de gelatina exerceram efeitos significativos (p<0,05) sobre a cor, volume, densidade e comportamento reológico dos produtos, exceto a atividade de água que foi influenciada apenas pelo primeiro fator. Sob a perspectiva reológica apenas a amostra com maior nível de substituição de gordura foi classificada como pseudoplástica. A gelatina contribuiu no aumento do teor proteico das formulações. A consistência foi significativamente (p<0,05) influenciada pelas propriedades da fase de gelatina, apresentando ampla faixa de plasticidade nas diferentes temperaturas (10, 20 e 30 °C). A consistência dos spreads aumentou em todas as temperaturas durante os 90 dias de armazenamento. Observou-se por meio da análise sensorial que os fatores em estudo provocaram alterações nos atributos sensoriais do produto suficiente para a percepção dos provadores. A formulação com 75 % de substituição de gordura seguida pelas formulações com 50 % e 0,8 % de concentração de gelatina apresentaram a maior aceitabilidade. Com o máximo de substituição de gordura chegou-se a 53 % de redução de custos. Contudo, os resultados obtidos sugerem que a aplicação de gelatina de pés de frango pode ser vantajosa, já que contribuiu com a formulação de spreads de chocolate menos calóricos e com propriedades físico-químicas e sensoriais adequadas para uma possível comercialização.

O efeito de diferentes volumes de treinamento de força nas adaptações funcionais e morfológicas da musculatura esquelética em indivíduos treinados

O objetivo desse estudo foi verificar o efeito de diferentes volumes de treinamento de força na força máxima de membros inferiores e na hipertrofia do reto femoral e do vasto lateral após quatro, oito e doze semanas em indivíduos treinados em força. Vinte e seis indivíduos jovens saudáveis do sexo masculino (idade 23,6 ± 4,6 anos, massa corporal 76,6 ± 7,5 kg, estatura 1,75 ± 0,1 cm), com tempo médio de treinamento de força (4,7 ± 4,1 anos) foram divididos em três grupos experimentais, treinamento de força alto volume (TFAV, n = 8), treinamento de força médio volume (TFMV, n = 9) e treinamento de força baixo volume (TFBV, n = 9). As medidas de força dinâmica máxima (1RM) e de área de secção transversa muscular (ASTM) do reto femoral (RF) e do vasto lateral (VL) foram realizadas nos momentos pré- treinamento, pós quatro semanas, pós oito semanas e pós-treinamento. O volume total de treinamento apresentou aumento estatístico para todos os grupos TFAV (p < 0,0001), TFMV (p < 0,0001) e TFBV (p < 0,0001) ao longo do período experimental. Os valores de 1RM aumentaram de maneira significativa após a oitava semana de treinamento TFAV (11,8 ± 4,7%; p < 0,0001) e TFMV (12,1 ± 8,5%; p < 0,0001) e TFBV (9,6 ± 7,3%; p < 0,001) e no pós-treinamento TFAV (13,9 ± 3,9%; p < 0,0001), TFMV (16,7 ± 10,8%; p < 0,0001) e TFBV (14,0 ± 8,1%; p < 0,0001) para todos os grupos, porém não foi observado diferença entre os grupos. A ASTM do RF apresentou aumento estatístico no pós-treinamento somente para o grupo TFAV (15,0 ± 11,9%; p < 0,0001). Apenas o grupo TFAV aumentou estatisticamente a ASTM do VL após quatro semanas de treinamento (7,71 ± 4,42%; p < 0,0001), porém todos os grupos aumentaram significativamente a ASTM do VL após oito semanas de treinamento TFAV (11,37 ± 3,88%; p < 0,0001), TFMV (9,68 ± 9,36%; p < 0,0001) e TFBV (7,26 ± 3,15%; p < 0,01) e no pós-treinamento TFAV (14,54 ± 4,07%; p < 0,0001), TFMV (14,77 ± 8,24%; p < 0,0001) e TFBV (8,66 ± 3,97%; p < 0,001), porém não foi observado diferença entre os grupos. Os resultados do presente estudo demonstraram que, independente do volume adotado, os ganhos de força máxima foram semelhantes. Por outro lado, a ASTM foi influenciada pelo volume de treinamento, dado que o grupo TFAV foi o único que apresentou aumento significativo da ASTM do RF no pós-treinamento e aumentou a ASTM do VL com apenas quatro semanas de treinamento

Tratamento superficial dos agregados reciclados de RCD com hidrofugantes.

A proposta busca reduzir a absorção de água dos agregados reciclados de Resíduos de Construção e Demolição (RCD), através do tratamento de sua superfície com hidrofugante, de maneira que estes não aumentem o consumo de água tampouco reduzam a resistência mecânica. Os agregados reciclados de RCD foram caracterizados quanto à granulometria, morfologia, porosidade, ângulo de contato aparente, absorção de água, análise de imagem e rugosidade superficial. Em seguida, os mesmos foram tratados superficialmente com dois tipos de hidrofugante: solução de silano e parafina. Todos os tratamentos permitiram reduzir a absorção de água dos agregados reciclados de RCD. O tratamento por imersão foi aquele que permitiu obter os melhores resultados e a menor variabilidade entre as técnicas. Os tratamentos hidrofugam a superfície, apesar da parafina apresentar maior molhabilidade. O tratamento com parafina apresentou, em termos de absorção de água, resultados inferiores e menos variáveis que o silano. Os parâmetros de rugosidade constataram que, ambos os materiais perdem seu perfil topográfico original quando tratados com parafina. Os diferentes agregados reciclados com cada hidrofugante foram aplicados em materiais cimentícios analisados no estado fresco e no estado endurecido. Os tratamentos reduziram a absorção e o consumo de água de mistura, porém afetaram negativamente as propriedades mecânicas, devido nova interface gerada (no caso específica de parafina) e falta de molhabilidade nas superfícies.

Strain and damage sensing properties on multifunctional cement composites with CNF admixture

Both strain and damage sensing properties on carbon nanofiber cement composites (CNFCC) are reported in the present paper. Strain sensing tests were first made on the material’s elastic range. The applied loading levels have been previously calculated from mechanical strength tests. The effect of several variables on the strain-sensing function was studied, e.g. cement pastes curing age, current density, loading rate or maximum stress applied. All these parameters were discussed using the gage factor as reference. After this first set of elastic experiments, the same specimens were gradually loaded until material’s failure. At the same time both strain and resistivity were measured. The former was controlled using strain gages, and the latter using a multimeter on a four probe setup. The aim of these tests was to prove the sensitivity of these CNF composites to sense their own damage, i.e. check the possibility of fabricating structural damage sensors with CNFCC’s. All samples with different CNF dosages showed good strain-sensing capacities for curing periods of 28 days. Furthermore, a 2%CNF reinforced cement paste has been sensitive to its own structural damage.

Influencia de diferentes condiciones de curado en la estructura porosa y en las propiedades a edades tempranas de morteros que contienen ceniza volante y escoria de alto horno

En este trabajo se ha estudiado la evolución de la microestructura, propiedades de durabilidad y resistencias mecánicas de morteros preparados con cementos comerciales, que contienen ceniza volante (entre un 21% y un 35%) y escoria de alto horno (entre un 66% y un 80%), expuestos a tres ambientes, un ambiente óptimo de laboratorio, y dos ambientes representativos del clima Atlántico y Mediterráneo respectivamente. Como referencia de comportamiento, también se ensayaron morteros de cemento Portland. La microestructura se caracterizó mediante porosimetría de intrusión de mercurio. En lo referente a la durabilidad, se estudiaron los coeficientes de absorción capilar y de migración de cloruros en estado no estacionario. También se determinó la resistencia a compresión de los morteros. Los ensayos se realizaron a 7, 28 y 90 días. La principal conclusión alcanzada es que los cementos con cenizas y escorias expuestos a condiciones ambientales representativas de los climas Atlántico y Mediterráneo, pueden desarrollar unas propiedades en servicio adecuadas al cabo de tres meses.

Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties

A heterofunctional support for enzyme immobilization may be defined as that which possesses several distinct functionalities on its surface able to interact with a protein. We will focus on those supports in which a final covalent attachment between the enzyme and the support is achieved. Heterofunctionality sometimes has been featured in very old immobilization techniques, even though in many instances it has been overlooked, giving rise to some misunderstandings. In this respect, glutaraldehyde-activated supports are the oldest multifunctional supports. Their matrix has primary amino groups, the hydrophobic glutaraldehyde chain, and can covalently react with the primary amino groups of the enzyme. Thus, immobilization may start (first event of the immobilization) via different causes and may involve different positions of the enzyme surface depending on the activation degree and immobilization conditions. Other “classical” heterofunctional supports are epoxy commercial supports consisting of reactive covalent epoxy groups on a hydrophobic matrix. Immobilization is performed at high ionic strength to permit protein adsorption, so that covalent attachment may take place at a later stage. Starting from these old immobilization techniques, tailor-made heterofunctional supports have been designed to permit a stricter control of the enzyme immobilization process. The requirement is to find conditions where the main covalent reactive moieties may have very low reactivity toward the enzyme. In this Review we will discuss the suitable properties of the groups able to give the covalent attachment (intending a multipoint covalent attachment), and the groups able to produce the first enzyme adsorption on the support. Prospects, limitations, and likely pathways for the evolution (e.g., coupling of site-directed mutagenesis and thiol heterofunctional supports of enzyme immobilization on heterofunctional supports) will be discussed in this Review.

Activated Carbon Fibre Monoliths for Hydrogen Storage

Porous adsorbents are currently investigated for hydrogen storage application. From a practical point of view, in addition to high porosity developments, high material densities are required, in order to confine as much material as possible in a tank device. In this study, we use different measured sample densities (tap, packing, compacted and monolith) for analyzing the hydrogen adsorption behavior of activated carbon fibres (ACFs) and activated carbon nanofibres (ACNFs) which were prepared by KOH and CO2 activations, respectively. Hydrogen adsorption isotherms are measured for all of the adsorbents at room temperature and under high pressures (up to 20 MPa). The obtained results confirm that (i) gravimetric H2 adsorption is directly related to the porosity of the adsorbent, (ii) volumetric H2 adsorption depends on the adsorbent porosity and importantly also on the material density, (iii) the density of the adsorbent can be improved by packing the original adsorbents under mechanical pressure or synthesizing monoliths from them, (iv) both ways (packing under pressure or preparing monoliths) considerably improve the storage capacity of the starting adsorbents, and (v) the preparation of monoliths, in addition to avoid engineering constrains of packing under mechanical pressure, has the advantage of providing high mechanical resistance and easy handling of the adsorbent.

Comparison Between Dcrit Considering the Abrupt Variation and Inflexion in the Concrete Mercury Intrusion Porosimetry Curve

Mercury intrusion porosimetry (MIP) has been widely used to evaluate the quality of concrete through the pore size distribution parameters. Two of these parameters are the critical pore diameter (Dcrit) and the percentage of the most interconnected net of pores compared to the total volume of pores. Some researchers consider Dcrit as the diameter obtained from the inflexion point of the cumulative mercury intrusion curve while others consider Dcrit as the diameter obtained from the point of abrupt variation in the same curve. This study aims to analyze two groups of concretes of varying w/c ratios, one cast with pozzolanic cement and another with high initial strength cement, in order to determine which of these diameters feature a better correlation with the quality parameters of the concretes. The concrete quality parameters used for the evaluations were (1) the w/c ratios and (2) chloride diffusion coefficients measured at approximately 90 days. MIP cumulative distributions of the same concretes were also measured at about 90 days, and Dcrit values were determined (1) from the point of abrupt variation and alternatively, (2) from the inflexion point of each of these plots. It was found that Dcrit values measured from the point of abrupt variation were useful indicators of the quality of the concrete, but the Dcrit values based on the inflexion points were not. Hence, it is recommended that Dcrit and the percentage of the most interconnected volume of pores should be obtained considering the point of abrupt variation of the cumulative curve of pore size distribution.

Petrology, geochemistry and K-Ar Age determination of De Gerlache Seamount basalts

The De Gerlache Seamounts are two topographic highs in the Bellingshausen Sea, southeastern Pacific. Petrological and geochemical studies together with K-Ar age determinations were carried out on four dredged basalt samples collected during a RV Polarstern expedition (ANT-XII/4) in 1995. Minor and trace element composition suggest alkaline basalt compositions. Compared to alkaline basalts of adjacent West Antarctica (the Jones Mountains) and of Peter I Island, the samples have lower mg-numbers, lower Ni and Cr contents and lower high field-strength elements (HFSE)/Nb and large-ion lithophile elements (LILE)/HFSE ratios. Three of the four samples have low K, Rb, and Cs concentrations relative to alkaline basalts. The K-depletion and other elemental concentrations may be explained by 1.1% melting of amphibole bearing mantle material. Additionally, low Rb and Ba values suggest low concentrations of these elements in the mantle source. K-Ar age determinations yield Miocene ages (20-23 Ma) that are similar in age to other alkaline basalts of West Antarctica (Thurston Island, the Jones Mountains, Antarctic Peninsula) and the suggested timing of onset of Peter I Island volcanism (~10-20 Ma). The occurrence of the DGS and Peter I Island volcanism along an older but reactivated tectonic lineation suggests that the extrusions exploited a zone of pre-existing lithospheric weakness. The alkaline nature and age of the DGS basalts support the assumption of plume activity in the Bellingshausen Sea.