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Dos Anjos L.C.,University of Brasilia | Gomes F.M.M.,University of Brasilia | Do Couto L.L.,University of Brasilia | Mourao C.A.,University of Brasilia | And 4 more authors.
Life Sciences | Year: 2016

Anxiety disorders are major health problems in terms of costs stemming from sick leave, disabilities, healthcare and premature mortality. Despite the availability of classic anxiolytics, some anxiety disorders are still resistant to treatment, with higher rates of adverse effects. In this respect, several toxins isolated from arthropod venoms are useful in identifying new compounds to treat neurological disorders, particularly pathological anxiety. Thus, the aims of this study were to identify and characterize an anxiolytic peptide isolated from the venom of the social wasp Polybia paulista. The peptide was identified as Polisteskinin R, with nominal molecular mass [M + H]+ = 1301 Da and primary structure consisting of Ala-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH. The anxiolytic effect was tested using the elevated plus maze test. Moreover, adverse effects on the spontaneous behavior and motor coordination of animals were assessed using the open field and rotarod tests. Polisteskinin R induced a dose-dependent anxiolytic effect. Animals treated with the peptide and diazepam spent significantly more time into the open arms when compared to the groups treated with the vehicle and pentylenetetrazole. No significant differences in spontaneous behavior or motor coordination were observed between the groups, showing that the peptide was well tolerated. The interaction by agonists in both known BK receptors induces a variability of physiological effects; Polisteskinin R can act on these receptors, inducing modulatory activity and thus, attenuating anxiety behaviors. The results of this study demonstrated that the compound Polisteskinin R exerted potent anxiolytic effects and its analogues are promising candidates for experimental pharmacology. © 2016 Elsevier Inc. Source

Carvalho J.O.,University of Sao Paulo | Silva L.P.,Laboratory of Mass Spectrometry | Sartori R.,University of Sao Paulo | Dode M.A.N.,Laboratory of Animal Reproduction | Dode M.A.N.,University of Brasilia
PLoS ONE | Year: 2013

Sperm dimensions and the question of whether X and Y chromosome-bearing sperm differ in size or shape has been of great interest, especially for the development of alternative methods to sort or classify sperm cells. The aim of the present study was to evaluate possible differences in the shape and size of the sperm head between X and Y chromosome-bearing sperm by atomic force microscopy (AFM). One ejaculate per bull (n = 4) was used. Each ejaculate was separated into four fractions: non-sexed (NS), sexed for X-sperm (SX), sexed for Y-sperm (SY) and a pooling of SX and SY samples (SXY). Using AFM, 400 sperm heads per group were measured. Twenty three structural features were assessed including one-, two- and three-dimensional parameters and shape descriptors. These measurements determine the micro- to nanoscale features of X- and Y-bearing chromosomes in sperm cells. No differences were observed for any individual variables between SX and SY groups. Next, a simultaneous evaluation of all features using statistical discriminant analysis was performed to determine if it was possible to distinguish to which group belong each individual cells. This analysis clearly showed, a distinct separation of NS, SXY, SX and SY groups. The recognition of this structural possibility to distinguish between X and Y sperm cell might improve the understanding of sperm cells biology. These results indicated that the associations of several structural measurements of the sperm cell head are promising candidates for development of a new method of sperm sexing. © 2013 Carvalho et al. Source

von Gal Milanezi N.,University of Brasilia | Mendoza D.P.G.,University of Brasilia | de Siqueira F.G.,University of Brasilia | Silva L.P.,Laboratory of Mass Spectrometry | And 2 more authors.
Bioenergy Research | Year: 2012

Aspergillus niger van Tieghem LPM 93 was shown in an earlier study to produce the most thermostable β-xylanase, which was effective for improving brightness and delignification of non-delignified and oxygen-bleached samples of eucalyptus kraft pulp. Here, we report the production, purification, and characterization of a xylan-degrading enzyme (XynI) from this strain grown in submerged liquid cultivation on medium containing sugar cane bagasse as the carbon source. XynI was isolated by ultrafiltration and gel-filtration chromatography and characterized. The fungus displayed high levels of xylanolytic activity after the second day of cultivation, and this activity remained constant up to the 50th day. The molecular mass of XynI was in the range of 32-33 kDa as determined by mass spectrometry and SDS-PAGE. The two-dimensional gel electrophoresis analysis showed the existence of multiple forms of β-xylanases in XynI. XynI showed the highest activity at 50°C and pH 4.5 and was stable in sodium acetate buffer at pH 4.5. The K m and V max values were 47.08 mg/ml and 3.02 IU/ml, respectively. Salts inhibited the activity of XynI to different degrees. N-Bromosuccinimide caused marked inhibition of XynI. On the other hand, β-mercaptoethanol and l-tryptophan were the best enzyme activators. © 2011 Springer Science+Business Media, LLC. Source

News Article
Site: http://phys.org/biology-news/

Now, for the first time, a team of researchers from The Rockefeller University, Brookhaven National Laboratory, and Stony Brook University has revealed that vital complex's molecular architecture. And to their surprise, it does not look as they had expected. "Our finding goes against decades of textbook drawings of what people thought the replisome should look like," says Michael O'Donnell, Anthony and Judith Evnin Professor, head of Rockefeller's Laboratory of DNA Replication and a Howard Hughes Medical Institute investigator. He is the senior author of the study, published November 2 in Nature Structural and Molecular Biology. "However, it's a recurring theme in science that nature does not always turn out to work the way you thought it did." The findings focus on the replisome found in eukaryotic organisms, a category that includes a broad swath of living things, including humans and other multicellular organisms. O'Donnell and others have long studied replisomes in simpler, single-celled bacteria, but the more complex version has over 30 different gears, or proteins, and required about 15 years for O'Donnell's lab to obtain. Through these previous studies, his lab has learned how to assemble the more complex replisome from its components. But until now, no pictures existed to show just how everything fit together in the eukaryotic replisome. To create them, the team began building the complete structure piece by piece, and examining its shape in the electron microscope—a powerful device used to study protein structures, and a specialty of co-author Huilin Li, a molecular biologist at Brookhaven National Laboratory and Stony Brook University. The pictures Li and members of his lab captured were the first ever made of a complete replisome from any type of cell. The DNA helix has two DNA strands, and each is duplicated by a separate DNA polymerase, an enzyme that creates DNA molecules by pairing single nucleotides, the basic units of DNA, with their matching partners. Another enzyme in the replisome is the helicase that, like a zipper, is responsible for separating DNA into two single strands in preparation for replication. For years, the two polymerases were thought to follow along behind the helicase, or below it, as it unzips the strands. But the new pictures of the replisome showed that one polymerase sits above the helicase. To identify which polymerase was at the top of the helicase, the team enlisted the help of co-authors postdoc Yi Shi and Brian Chait, the Camille and Henry Dreyfus Professor at Rockefeller and head of the Laboratory of Mass Spectrometry and Gaseous Ion Chemistry. They identified the top polymerase as Pol-ε. Why the eukaryotic replisome developed such a structure is not known. O'Donnell and his colleagues suspect that it may have something to do with the evolution of multicellularity. As the helicase unzips two strands of DNA, it encounters nucleosomes, particles that tightly bundle DNA to fit it into a cell's nucleus. These must be dislodged and then distributed to one new strand or the other. Previous work has shown that Pol-ε binds nucleosomes, and it may be that while riding atop the helicase, Pol-ε is in charge of distributing nucleosomes to the two strands, O'Donnell suggests. "Changes to nucleosomes carry epigenetic information that instructs different cells to become the different tissues of the body, such as heart, brain, and other organs during embryonic development," O'Donnell says. "So we can speculate that Pol-ε's interaction with nucleosomes could play a role in assigning different epigenetic identities to the two new daughter cells after cell division, that instruct them to form different organs during development of a multicellular animal." More information: The architecture of a eukaryotic replisome, Nature Structural and Molecular Biology, DOI: 10.1038/nsmb.3113

News Article
Site: http://phys.org/biology-news/

Michael Rout, head of the Laboratory of Cellular and Structural Biology, and Brian Chait, head of the Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, together with their colleagues, have been working to understand the nuclear pore complex for nearly two decades. They first isolated and described the components of the yeast nuclear pore complex in 2000 and then released a first draft of its structure in 2007. In yeast, as well as in humans, the nuclear pore complex is composed of an inner ring sandwiched between two outer rings: one facing in and one facing out. Attached to these outer rings are classes of proteins that are compatible with the unique chemistry of either the nucleus or cytoplasm found in the rest of the cell. This asymmetric distribution of nuclear pore components is important for helping to establish the directionality of transport through the pore. The complex is so fundamental that its architecture was thought to be shared by all eukaryotes. However, Samson Obado, a research associate in the Rout lab, has found a eukaryotic species that has pores with a structure unlike any that has yet been studied, a finding which implies that the pore's evolution is probably more involved than had previously been assumed. Obado's results were published in February in PLoS Biology. "We were driven by curiosity about the evolution of the nuclear pore complex," says Obado. Almost everything we know about the complex is from yeast and human, which although quite far apart in evolutionary terms are nevertheless closely related when compared to plants and many other single-celled eukaryotes. "But what did the original pores look like, and how have they developed in different eukaryotes?" Obado wondered. He was the perfect candidate to investigate this, as his graduate studies focused on trypanosomes; these parasites, responsible for serious diseases such as sleeping sickness and Chagas disease, diverged from the families that developed into yeast, humans, plants, and other eukaryotes roughly one and a half billion years ago - close to the time of the last common ancestor of all eukaryotes. "Compared with many other eukaryotes, trypanosomes have an unusual and quirky molecular biology. A key example is how they transcribe their genes into messenger RNAs for translation into proteins, which is very different from textbook models. Furthermore, because they are so divergent, you can't just search for gene sequences similar to those in yeast or humans," Obado says. Rout, Chait, and colleagues worked together with Mark Field of Dundee University in Scotland to identify putative nuclear pore components from trypanosomes. To do so, the team walked protein by protein through the nuclear pore complex, purifying each one along with other proximal proteins. These other proteins, they figured, could be part of the nuclear pore complex as well. Obado then purified the new proteins to determine whether they were also part of the complex, along with their proximal proteins, and so on until he had a complete survey of all the proteins involved. Finally, they worked with the university's Electron Microscopy Resource Center to determine where key components are located with respect to each other. The result was a complete first picture of the entire trypanosome pore structure. The teams' results show that the architecture of the inner ring of the nuclear pore complex is fundamentally similar in trypanosomes, yeast, plants, and vertebrates, suggesting an ancient origin for this common feature. In contrast, the trypanosome nuclear pore complex possesses a unique mechanism tethering it to the surrounding nuclear envelope, and the outer ring is less well "conserved" by evolution, with additional never-before-seen components. However, the most notable difference between trypanosome nuclear pore complexes and others that have been examined to date, is that the trypanosome's nuclear pore complex exhibits a near-complete symmetry of its components, and lacks almost all the proteins in yeast and humans that are required to establish an asymmetric assembly within the pore. Instead, it seems that soluble proteins in the nucleus and cytoplasm are responsible for setting transport's directionality in these parasites for both proteins and RNA. The fact that the trypanosome pore has such dissimilarities with the pore of humans may provide an opportunity, says Obado. "These differences may offer something we can target therapeutically without risking harm to our own transport mechanisms." These discoveries also lend further credence to an idea in evolutionary theory first suggested by Rout and his colleagues. The theory, known as the "protocoatomer hypothesis," suggest that the nuclear pore and nuclear envelope share a common ancestor with structures that form coats on other membrane-bound structures in the cell, like those in the Golgi apparatus and endoplasmic reticulum. Indeed, the trypanosome nuclear pore components, despite their dissimilarities from other eukaryotes, still retain the kinds of structures and organization found in these other coats, the scientists say. Moreover, since the inner core of the trypanosome nuclear pore complex are structurally well preserved and similar to those from other eukaryotes, Rout and his team believe this core represents the original units of a simpler nuclear pore complex that pre-dates the last ancestor of all eukaryotes, and thus may provide new clues as to how the nucleus originally evolved. Explore further: Research suggests core nuclear pore elements shared by all eukaryotes More information: Samson O. Obado et al. Interactome Mapping Reveals the Evolutionary History of the Nuclear Pore Complex, PLOS Biology (2016). DOI: 10.1371/journal.pbio.1002365

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