Monitoring the positioning of short polycationic peptides in model lipid bilayers by combining hydrogen/deuterium exchange and electrospray ionization mass spectrometry

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

Profiling the proteome complement of the secretion from hypopharyngeal gland of Africanized nurse-honeybees (Apis mellifera L.)

The protein complement of the secretion from hypopharyngeal gland of nurse-bees (Apis mellifera L.) was partially identified by using a combination of 2D-PAGE, peptide sequencing by MALDI-PSD/MS and a protein engine identification tool applied to the honeybee genome. The proteins identified were compared to those proteins already identified in the proteome complement of the royal jelly of the honey bees. The 2D gel electrophoresis demonstrated this protein complement is constituted of 61 different polypepides, from which 34 were identified as follows: 27 proteins belonged to MRJPs family, 5 proteins were related to the metabolism of carbohydrates and to the oxido-reduction metabolism of energetic Substrates, I protein was related to the accumulation of iron in honeybee bodies and I protein may be a regulator of MRJP-1 oligomerization. The proteins directly involved with the carbohydrates and energetic metabolisms were: alpha glucosidase, glucose oxidase and alpha amylase, whose are members of the same family of enzymes, catalyzing the hydrolysis of the glucosidic linkages of starch; alcohol dehydrogenase and aldehyde dehydrogenase, whose are constituents of the energetic metabolism. The results of the present manuscript support the hypothesis that the most of these proteins are produced in the hypoharyngeal gland of nurse-bees and secreted into the RJ. (C) 2004 Elsevier Ltd. All rights reserved.

Planting of fungus onto hibernating workers of the fungus-growing ant Mycetosoritis clorindae (Attini, Formicidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Nesting biology and fungiculture of the fungus-growing ant, Mycetagroicus cerradensis: New light on the origin of higher-attine agriculture

The genus Mycetagroicus is perhaps the least known of all fungus-growing ant genera, having been first described in 2001 from museum specimens. A recent molecular phylogenetic analysis of the fungus-growing ants demonstrated that Mycetagroicus is the sister to all higher attine ants (Trachymyrmex, Sericomyrmex, Acromyrmex, Pseudoatta, and Atta), making it of extreme importance for understanding the transition between lower and higher attine agriculture. Four nests of Mycetagroicus cerradensis near Uberlandia, Minas Gerais, Brazil were excavated, and fungus chambers for one were located at a depth of 3.5 meters. Based on its lack of gongylidia (hyphal-tip swellings typical of higher attine cultivars), and a phylogenetic analysis of the ITS rDNA gene region, M. cerradensis cultivates a lower attine fungus in Clade 2 of lower attine (G3) fungi. This finding refines a previous estimate for the origin of higher attine agriculture, an event that can now be dated at approximately 21-25 mya in the ancestor of extant species of Trachymyrmex and Sericomyrmex.

Morphological architecture of Actinocephalus (Koern.) sano (Eriocaulaceae-Poales)

Actinocephalus exhibits perhaps more diversity in habit than any other genus of Eriocaulaceae. This variation is largely a result of differences in the arrangement of the paraclades. Based on the analysis of stem architecture of all 25 species of Actinocephalus, the following patterns were established: (1) leaf rosette, with no elongated axis, instead the axillary paraclades originating directly from the short aerial stem, (2) rosette axis continuing into an elongated axis with spirally arranged paraclades, (3) an elongated axis originating from a rhizome, with ramified paraclades, and (4) an elongated axis originating from a short aerial stem, with paraclades arranged in a subwhorl. The elongated axis exhibits indeterminate growth only in pattern 4. Patterns 3 and 4 are found exclusively in Actinocephalus; pattern I occurs in many other genera of Eriocaulaceae, while pattern 2 is also found in Syngonanthus and Paepalanthus. Anatomically, each stem structure (i.e., paraclade, elongated axis, short aerial stem, rhizome) is thickened in a distinctive way and this can be used to distinguish them. Specifically, elongated axes and paraclades lack thickening, thickening of short aerial stems results from the primary thickening meristem and/or the secondary thickening meristem. Thickening of rhizomes results from the activity of the primary thickening meristem. (c) 2008 Elsevier GmbH. All rights reserved.

Nest architecture of Pachycondyla striata Fr. Smith, 1858 (Formicidae, Ponerinae)

Nine nests of Pachycondyla striata Fr. Smith, 1858 were excavated between 2006 and 2007 at the UNESP university campus in Rio Claro, SP, southeastern Brazil. The P. striata population was larger in the two nests excavated in the months of August and November, 2006. The nests of this species are simple and poorly elaborated, with chambers and tunnels close to the ground surface. P. striata places the pupae in chambers isolated from the other individuals. The lack of chambers with the specific purpose of conditioning food and garbage underscores the simplicity of these nests.

Association of the stilt bug Jalysus ossesae Henry (Hemiptera : Heteroptera : Berytidae) with myrmecophytic plants of the genus Maieta (Melastomataceae) in an upland forest area in central Amazon, Brazil

Maieta guianensis Aubl. and M. poeppigii Mart. ex. Triana (Melastomataceae) are among the most common myrmecophytic plants in the Amazonian forest understory. These myrmecophytes are colonized exclusively by the ants Pheidole minutula Mayr or Crematogaster sp. and usually host two other arthropods, the spider Faiditus subflavus Exline and Levi and the recently described stilt bug Jalysus ossesae Henry. In this study, the association between J ossesae and the myrmecophytic plants M. guianensis and M. poeppigii in an upland forest area in central Amazon, Brazil, is described. The presence of the stilt bugs on M. guianensis and M. poeppigii and on plants around these myrmecophytes was recorded in five transects. The number and position of the stilt bugs on the leaf surface (upper or lower) and leaf type (with or without domatia) of these myrmecophytes, as well as their behavioral acts, were recorded. Jalysus ossesae was found only on the myrmecophytic plants M. guianensis and M. poeppigii. The stilt bug occurred at similar frequencies on M. guianensis and M. poeppigii, and the number of leaves significantly influenced the presence and number of stilt bugs on these myrmecophytes. Feeding, agonistic interaction between males, and mating were observed. Our data indicate that J. ossesae uses the myrmecophytes M. guinanensis and M. poeppigii as reproductive and foraging sites.

Fundamental Limits on Wavelength, Efficiency and Yield of the Charge Separation Triad

In an attempt to optimize a high yield, high efficiency artificial photosynthetic protein we have discovered unique energy and spatial architecture limits which apply to all light-activated photosynthetic systems. We have generated an analytical solution for the time behavior of the core three cofactor charge separation element in photosynthesis, the photosynthetic cofactor triad, and explored the functional consequences of its makeup including its architecture, the reduction potentials of its components, and the absorption energy of the light absorbing primary-donor cofactor. Our primary findings are two: First, that a high efficiency, high yield triad will have an absorption frequency more than twice the reorganization energy of the first electron transfer, and second, that the relative distance of the acceptor and the donor from the primary-donor plays an important role in determining the yields, with the highest efficiency, highest yield architecture having the light absorbing cofactor closest to the acceptor. Surprisingly, despite the increased complexity found in natural solar energy conversion proteins, we find that the construction of this central triad in natural systems matches these predictions. Our analysis thus not only suggests explanations for some aspects of the makeup of natural photosynthetic systems, it also provides specific design criteria necessary to create high efficiency, high yield artificial protein-based triads.

A systematic approach to identify STRE-binding proteins of the gsn glycogen synthase gene promoter in Neurospora crassa

The gene encoding glycogen synthase in Neurospora crassa (gsn) is transcriptionally down-regulated when mycelium is exposed to a heat shock from 30 to 45 degrees C. The gsn promoter has one stress response element (STRE) motif that is specifically bound by heat shock activated nuclear proteins. In this work, we used biochemical approaches together with mass spectrometric analysis to identify the proteins that bind to the STRE motif and could participate in the gsn transcription regulation during heat shock. Crude nuclear extract of heat-shocked mycelium was prepared and fractionated by affinity chromatography. The fractions exhibiting DNA-binding activity were identified by electrophoretic mobility shift assay (EMSA) using as probe a DNA fragment containing the STRE motif DNA-protein binding activity was confirmed by Southwestern analysis. The molecular mass (MM) of proteins was estimated by fractionating the crude nuclear extract by SDS-PAGE followed by EMSA analysis of the proteins corresponding to different MM intervals. Binding activity was detected at the 30-50 MM kDa interval. Fractionation of the crude nuclear proteins by IEF followed by EMSA analysis led to the identification of two active fractions belonging to the pIs intervals 3.54-4.08 and 6.77-7.31. The proteins comprising the MM and pI intervals previously identified were excised from a 2-DE gel, and subjected to mass spectrometric analysis (MALDI-TOF/TOF) after tryptic digestion. The proteins were identified by search against the MIPS and MIT N. crassa databases and five promising candidates were identified. Their structural characteristics and putative roles in the gsn transcription regulation are discussed.

Formation of TiO2 ceramic foams from the integration of the sol-gel method with surfactants assembly and emulsion

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Study of the proteins in the defatted flour and protein concentrate of baru nuts (Dipteryx alata Vog)

O baru (Dipteryx alata Vog.) é uma leguminosa abundante no Cerrado brasileiro, cuja castanha pode ser explorada através do uso sustentável para o aproveitamento das frações proteicas e lipídicas. Este trabalho teve como objetivo estudar as proteínas desta castanha, presentes na farinha desengordurada e no concentrado proteico, quanto as suas propriedades funcionais, ao perfil das frações proteicas e à digestibilidade in vitro. A farinha desengordurada com hexano foi submetida à extração no pH de maior solubilidade das proteínas, obtendo-se o concentrado proteico. O perfil eletroforético das frações proteicas foi avaliado em gel de SDS-PAGE. As propriedades funcionais indicaram a possibilidade de emprego em diversos alimentos, assim como a soja, conferindo capacidade de absorção de água, capacidade de absorção de óleo, propriedades emulsificantes e espumabilidade. As globulinas, seguidas das albuminas, são as frações majoritárias da farinha e do concentrado proteico, respectivamente. A digestibilidade foi superior no concentrado em relação à farinha desengordurada.

Purification and properties of acid phosphatase (EC 3.1.3.2) secreted by strain 74A of the mould Neurospora crassa

Both P-i-repressible acid phosphatases, IIb (mycelial) and IIc (extracellular), synthesized by Neurospora crassa and purified to apparent homogeneity by 7.5% PAGE, are monomers, are inhibited by 2 mM ZnCl2 and are nonspecifically stimulated by salts. However, the IIc form is activated by p-nitrophenylphosphate (in a negative cooperativity effect with a K-0.5 of 2.5 mM) whereas form IIb shows Michaelis kinetics, with a K-m of 0.5 mM. Thus, since both enzymatic forms may be expressed by the same gene (pho-3), it is possible that post-translational modifications lead to the excretion of an enzymatic form with altered Michaelis kinetics compared with the enzymatic form retained by the mycelium.

Nest architecture of Atta laevigata (F. Smith, 1858) (Hymenoptera : Formicidae)

The internal structure of three nests of Atta laevigata was studied in Botucatu city, São Paulo State, Brazil. Two nests were excavated through the opening of trenches and one was cement-moulded to enable a better view of the internal structures. The three nests presented a similar structure, with the depths and number of chambers varying as a function of the loose soil area of the nests. The number of chambers ranged from 1149 to 7864, reaching as deep as 7 m underground. Chamber volume ranged from 0.03 to 511. The foraging tunnels extended as far as 70m from the loose soil region. The nests of this species are the deepest within the genus Atta and have the highest number of chambers.

Cytoarchitecture and musculotopic organization of the facial motor nucleus in Cebus apella monkey

The architecture and musculotopic organization of the facial motor nucleus in the Cebus apella monkey (a New World primate) were investigated using histological techniques and a multiple labelling strategy, in which horseradish peroxidase-conjugated neuroanatomical tracers (CTB-HRP and WGA-HRP) and fluorescent tracers were injected into individual facial muscles. The facial motor nucleus was formed by multipolar motoneurons and had an ovoid shape, with its rostrocaudal axis measuring on average 1875 mum. We divided the nucleus into four different subnuclei: medial, intermediate, dorsal and lateral. Retrograde labelling patterns revealed that individual muscles were innervated by longitudinal functional columns of motoneurons. The columns of the orbicularis oculi, zygomaticus, orbicularis oris, auricularis superior, buccinator and platysma muscles were located in the dorsal, intermediate, lateral, medial, lateral and intermediate subnuclei, respectively. However, the motoneuron columns of the levator labii superioris alaeque nasi muscle and frontalis muscle could not be associated with a specific subnucleus. The present results confirm previous studies regarding the musculotopic organization of the facial motor nucleus. However, we observed some particularities in terms of the relative size of each column in C. apella, which might be related to the functional and behavioral importance of each muscle in the particular context of this primate.

A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: Biochemical characterization, crystallization, myotoxic and edematogenic activity

A, M. Soares, V, M, Rodrigues, M. I. Homsi-Brandeburgo, M. H. Toyama, F, R, Lombardi, K. Arni and J. R, Giglio. A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: Biochemical characterization, crystallization, myotoxic and edematogenic activity. Toxicon 36, 503-514, 1998.-Bothrops moojeni snake venom was fractionated on a CM-Sepharose column which was previously equilibrated with 0.05 M ammonium bicarbonate buffer at pH 8.0 and subsequently eluted with an ammonium bicarbonate concentration gradient from 0.05 to 0.5 M at constant pH (8.0) and temperature (25 degrees C). The fraction which eluted last (M-VI) showed, after direct lyophilization, a single band by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE, indicating an approximate M,. of 14 000 and 77 000, in the presence and absence of dithiothreitol, respectively. Its amino acid composition revealed a high level of hydrophobic and basic amino acids as well as 13 half-cystine residues. Its isoelectric point and extinction coefficient (E-1.0cm(1.0mg/ml) at 278 nm and pH 7.0) were 8.2 and 1.170, respectively. M-VI was devoid of phospholipase A(2) (PLA(2)) activity on egg yolk, as well as of hemorrhagic, anticoagulant and coagulant activities, but could induce drastic necrosis on skeletal muscle fibres as well as rapid and transient edema on the rat paw. Its N-terminal sequence: SLFELGKMILQETGKNPAKSYGVYGCNCGVGGRGKPKDATDRCCYVHKCCYK.... revealed high homology with other Lys 49 PLA(2)-like myotoxins from other bothropic venoms. Orthorhombic crystals of M-VI? which diffracted to a maximal resolution of 1.6 Angstrom. were obtained and indicated the presence of a dimer in the asymmetrical unit. (C) 1998 Elsevier B.V. Ltd. All rights reserved.