Finding rigid bodies in protein structures: Application to flexible fitting into cryoEM maps

We present RIBFIND, a method for detecting flexibility in protein structures via the clustering of secondary structural elements (SSEs) into rigid bodies. To test the usefulness of the method in refining atomic structures within cryoEM density we incorporated it into our flexible fitting protocol (Flex-EM). Our benchmark includes 13 pairs of protein structures in two conformations each, one of which is represented by a corresponding cryoEM map. Refining the structures in simulated and experimental maps at the 5–15 Å resolution range using rigid bodies identified by RIBFIND shows a significant improvement over using individual SSEs as rigid bodies. For the 15 Å resolution simulated maps, using RIBFIND-based rigid bodies improves the initial fits by 40.64% on average, as compared to 26.52% when using individual SSEs. Furthermore, for some test cases we show that at the sub-nanometer resolution range the fits can be further improved by applying a two-stage refinement protocol (using RIBFIND-based refinement followed by an SSE-based refinement). The method is stand-alone and could serve as a general interactive tool for guiding flexible fitting into EM maps.     

Energy use by the mountain pine beetle (Coleoptera: Curculionidae: Scolytinae) for dispersal by flight

The mountain pine beetle Dendroctonus ponderosae Hopkins is a major native pest of Pinus Linnaeus (Pinaceae) in western North America. Host colonization by the mountain pine beetle is associated with an obligatory dispersal phase, during which beetles fly in search of a suitable host. Mountain pine beetles use stored energy from feeding in the natal habitat to power flight before host colonization and brood production. Lipids fuel mountain pine beetle flight, although it is not known whether other energy sources are also used during flight. In the present study, we compare the level of energy substrates, proteins, carbohydrates and lipids of individual mountain pine beetles flown on flight mills with unflown control beetles. We use a colorimetric method to measure the entire metabolite content of each individual beetle. The present study reveals that mountain pine beetles are composed of more protein and lipid than carbohydrate. Both female and male mountain pine beetles use lipids and carbohydrates as energy sources during flight. There is variation between sexes, however, in the energy substrates used for flight. Male mountain pine beetles use protein, in addition to lipids and carbohydrates, to fuel flight, whereas protein content is not different between flown and control females.     

Identification of arylsulfonamides as Aquaporin 4 inhibitors

Carbonic anhydrase inhibitors AZA, EZA, and 4-acetamidobenzsulfonamide were found to inhibit human AQP4-M23 mediated water transport by 80%, 68%, and 23%, respectively, at 20 microM in an in vitro functional assay. AZA was found to have an IC50 against AQP4 of 0.9 microM. Phloretin was inactive under the same conditions.     

The role of nuclear pores in gene regulation,development and disease

Nuclear‐pore complexes (NPCs) are large protein channels that span the nuclear envelope (NE), which is a double membrane that encloses the nuclear genome of eukaryotes. Each of the typically 2,000–4,000 pores in the NE of vertebrate cells is composed of multiple copies of 30 different proteins known as nucleoporins. The evolutionarily conserved NPC proteins have the well‐characterized function of mediating the transport of molecules between the nucleoplasm and the cytoplasm. Mutations in nucleoporins are often linked to specific developmental defects and disease, and the resulting phenotypes are usually interpreted as the consequences of perturbed nuclear transport activity. However, recent evidence suggests that NPCs have additional functions in chromatin organization and gene regulation, some of which might be independent of nuclear transport. Here, we review the transport‐dependent and transport‐independent roles of NPCs in the regulation of nuclear function and gene expression.     

Phylogeography of Y-chromosome haplogroup I reveals distinct domains of prehistoric gene flow in europe

To investigate which aspects of contemporary human Y-chromosome variation in Europe are characteristic of primary colonization, late-glacial expansions from refuge areas, Neolithic dispersals, or more recent events of gene flow, we have analyzed, in detail, haplogroup I (Hg I), the only major clade of the Y phylogeny that is widespread over Europe but virtually absent elsewhere. The analysis of 1,104 Hg I Y chromosomes, which were identified in the survey of 7,574 males from 60 population samples, revealed several subclades with distinct geographic distributions. Subclade I1a accounts for most of Hg I in Scandinavia, with a rapidly decreasing frequency toward both the East European Plain and the Atlantic fringe, but microsatellite diversity reveals that France could be the source region of the early spread of both I1a and the less common I1c. Also, I1b*, which extends from the eastern Adriatic to eastern Europe and declines noticeably toward the southern Balkans and abruptly toward the periphery of northern Italy, probably diffused after the Last Glacial Maximum from a homeland in eastern Europe or the Balkans. In contrast, I1b2 most likely arose in southern France/Iberia. Similarly to the other subclades, it underwent a postglacial expansion and marked the human colonization of Sardinia ~9,000 years ago.     

Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter

Protein kinase D (PKD) regulates many diverse cellular functions in response to diacylglycerol. To monitor PKD signaling in live cells, we generated a genetically encoded fluorescent reporter for PKD activity, DKAR (D kinase activity reporter). DKAR expressed in mammalian cells undergoes reversible fluorescence resonance energy transfer changes upon activation and inhibition of endogenous PKD. Surprisingly, we find that agonist-evoked activation of PKD is driven not only by diacylglycerol production, but by Ca(2+). Furthermore, elevation of intracellular Ca(2+), in the absence of any other stimulus, is sufficient to activate PKD. Concurrent imaging of Ca(2+), diacylglycerol, and PKD activity reveals that thapsigargin-mediated elevation of intracellular Ca(2+) is closely followed by a robust increase in diacylglycerol production, in turn followed by PKD activation. The Ca(2+)-induced production of diacylglycerol and accompanying PKD activation is dependent on phospholipase C activity. These data reveal that Ca(2+) is a major contributor to the initiation of PKD signaling through positive feedback regulation of diacylglycerol production, unveiling a new mechanism in PKD activation.     

Oxygenated bisabolane fucosides from Carthamus lanatus L

The aerial parts of Carthamus lanatus (Asteraceae) afforded four new oxygenated bisabolane fucosides, 10-hydroperoxy-bisabola-2,11-diene 7-O-beta-D-fucopyranoside, 11-hydro-peroxy-bisabola-2,9-diene 7-O-beta-D-fucopyranoside, 10-hydroxy-bisabola-2,11-diene 7-O-beta-D-fucopyranoside and 11-hydroxy-bisabola-2,9-diene 7-O-beta-D-fucopyranoside together with the known compounds a-bisabolol beta-D-fucopyranoside, asperuloside, sitosterol 3-O-beta-D-glucoside and stigmasterol 3-O-beta-D-glucoside. Asperuloside appears to be the second representative of the iridoid monoterpene group found in the plant family Asteraceae, which until recently was considered to lack iridoids. The main constituent a-bisabolol fucoside exhibited noticeable antibacterial and cytotoxic activities.     

Effects of buthionine sulfoximine nifurtimox and benznidazole upon trypanothione and metallothionein proteins in Trypanosoma cruzi

Proteins rich in sulfhydryl groups, such as metallothionein, are present in several strains of the parasite Trypanosoma cruzi, the etiological agent of Chagas' disease. Metallothionein-like protein concentrations ranged from 5.1 to 13.2 pmol/mg protein depending on the parasite strain and growth phase. Nifurtimox and benznidazole, used in the treatment of Chagas' disease, decreased metallothionein activity by approximately 70%. T. cruzi metallothionein was induced by ZnCl2. Metallothionein from T. cruzi was partially purified and its monobromobimane derivative showed a molecular weight of approximately 10,000 Da by SDS-PAGE analysis. The concentration of trypanothione, the major glutathione conjugate in T. cruzi, ranged from 3.8 to 10.8 nmol/mg protein, depending on the culture phase. The addition of buthionine sulfoximine to the protozoal culture considerably reduced the concentration of trypanothione and had no effect upon the metallothionein concentration. The possible contribution of metallothionein-like proteins to drug resistance in T. cruzi is discussed.