Formation of Raft-Like Assemblies within Clusters of Influenza Hemagglutinin Observed by MD Simulations

The association of hemagglutinin (HA) with lipid rafts in the plasma membrane is an important feature of the assembly process of influenza virus A. Lipid rafts are thought to be small, fluctuating patches of membrane enriched in saturated phospholipids, sphingolipids, cholesterol and certain types of protein. However, raft-associating transmembrane (TM) proteins generally partition into Ld domains in model membranes, which are enriched in unsaturated lipids and depleted in saturated lipids and cholesterol. The reason for this apparent disparity in behavior is unclear, but model membranes differ from the plasma membrane in a number of ways. In particular, the higher protein concentration in the plasma membrane may influence the partitioning of membrane proteins for rafts. To investigate the effect of high local protein concentration, we have conducted coarse-grained molecular dynamics (CG MD) simulations of HA clusters in domain-forming bilayers. During the simulations, we observed a continuous increase in the proportion of raft-type lipids (saturated phospholipids and cholesterol) within the area of membrane spanned by the protein cluster. Lateral diffusion of unsaturated lipids was significantly attenuated within the cluster, while saturated lipids were relatively unaffected. On this basis, we suggest a possible explanation for the change in lipid distribution, namely that steric crowding by the slow-diffusing proteins increases the chemical potential for unsaturated lipids within the cluster region. We therefore suggest that a local aggregation of HA can be sufficient to drive association of the protein with raft-type lipids. This may also represent a general mechanism for the targeting of TM proteins to rafts in the plasma membrane, which is of functional importance in a wide range of cellular processes.     

Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases

The N(6)-(isopentenyl)adenosine (i(6)A) modification of some tRNAs at position A37 is found in all kingdoms and facilitates codon-specific mRNA decoding, but occurs in different subsets of tRNAs in different species. Here we examine yeasts' tRNA isopentenyltransferases (i.e., dimethylallyltransferase, DMATase, members of the Δ(2)-isopentenylpyrophosphate transferase, IPPT superfamily) encoded by tit1(+) in Schizosaccharomyces pombe and MOD5 in Saccharomyces cerevisiae, whose homologs are Escherichia coli miaA, the human tumor suppressor TRIT1, and the Caenorhabditis elegans life-span gene product GRO-1. A major determinant of miaA activity is known to be the single-stranded tRNA sequence, A36A37A38, in a stem-loop. tRNA(Trp)(CCA) from either yeast is a Tit1p substrate, but neither is a Mod5p substrate despite the presence of A36A37A38. We show that Tit1p accommodates a broader range of substrates than Mod5p. tRNA(Trp)(CCA) is distinct from Mod5p substrates, which we sort into two classes based on the presence of G at position 34 and other elements. A single substitution of C34 to G converts tRNA(Trp)(CCA) to a Mod5p substrate in vitro and in vivo, consistent with amino acid contacts to G34 in existing Mod5p-tRNA(Cys)(GCA) crystal structures. Mutation of Mod5p in its G34 recognition loop region debilitates it differentially for its G34 (class I) substrates. Multiple alignments reveal that the G34 recognition loop sequence of Mod5p differs significantly from Tit1p, which more resembles human TRIT1 and other DMATases. We show that TRIT1 can also modify tRNA(Trp)(CCA) consistent with broad recognition similar to Tit1p. This study illustrates previously unappreciated molecular plasticity and biological diversity of the tRNA-isopentenyltransferase system of eukaryotes.     

Enzymes of aldoxime–nitrile pathway for organic synthesis

Aldoxime–nitrile pathway is one of the important routes of carbon and nitrogen metabolism in many life forms and a key interface for plant–microbe interactions. This pathway starts with transformation of amino acids to aldoximes, which are converted to nitriles and the later are ultimately hydrolyzed to acids and ammonia. Understanding and engineering of the enzymes involved in this pathway viz. cytochrome P450/CYP79, aldoxime dehydratase, nitrilase, nitrile hydratase, amidase and hydroxynitrile lyase, presents unprecedented opportunities in biocatalysis and green chemistry. Co-expressing these enzymes in prokaryotic and eukaryotic microbial hosts and tailoring their properties i.e. activity, specificity, stability and enantioselectivity may lead to develop sustainable bioprocesses for the synthesis of industrially important nitriles, amides and acids.     

Direct and indirect effects of zooplankton on algal composition in in situ grazing experiments

To examine both direct and indirect effects of macrozooplankton on phytoplankton species in Lake Biwa, we conducted in situ grazer-gradient experiments under different nutrient levels in summer, when Daphnia galeata dominated, and in autumn, when Eodiaptomus japonicus dominated. The experiments revealed that grazing pressure on phytoplankton was highly dependent on zooplankton species composition. Smaller phytoplankton species such as Stephanodiscus carconensis were more grazed when D. galeata was abundant, whereas large colonial diatom species such as Aulacoseira granulata were preferentially grazed when E. japonicus dominated. In addition, indirect effect of macrozooplankton through nutrient regeneration was suggested, although the magnitude of nutrient regeneration effects seemed to differ between D. galeata and E. japonicus. Specifically, growth rates of Sphaerocystis schroeteri were stimulated more by E. japonicus than by D. galeata. Macrozooplankton also enhanced the growth rates of colonial cyanobacteria such as Microcystis incerta, probably through decreasing the density of microzooplankton grazers (ciliates and rotifers). The results suggest that the effects of large zooplankton on phytoplankton populations are species-specific and cannot be understood without consideration of changes in abundance of other components of plankton communities.     

Energy generation from the CO oxidation-hydrogen production pathway in Rubrivivax gelatinosus

When incubated in the presence of CO gas, Rubrivivax gelatinosus CBS induces a CO oxidation-H2 production pathway according to the stoichiometry CO + H2O --> CO2 + H2. Once induced, this pathway proceeds equally well in both light and darkness. When light is not present, CO can serve as the sole carbon source, supporting cell growth anaerobically with a cell doubling time of nearly 2 days. This observation suggests that the CO oxidation reaction yields energy. Indeed, new ATP synthesis was detected in darkness following CO additions to the gas phase of the culture, in contrast to the case for a control that received an inert gas such as argon. When the CO-to-H2 activity was determined in the presence of the electron transport uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), the rate of H2 production from CO oxidation was enhanced nearly 40% compared to that of the control. Upon the addition of the ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), we observed an inhibition of H2 production from CO oxidation which could be reversed upon the addition of CCCP. Collectively, these data strongly suggest that the CO-to-H2 reaction yields ATP driven by a transmembrane proton gradient, but the detailed mechanism of this reaction is not yet known. These findings encourage additional research aimed at long-term H2 production from gas streams containing CO.     

Transfer of tuber soft rot and early blight resistances from<Emphasis Type="Italic"> Solanum brevidens</Emphasis> into cultivated potato

Tuber soft rot and early blight are serious potato diseases. Development of potato varieties resistant to these diseases has been hindered by the scarcity of resistant germplasm. A diploid wild species, Solanum brevidens, shows significant resistance to both diseases. Numerous potato breeding lines have been developed from a potato-S. brevidens somatic hybrid, A206. A BC₃ clone, C75-5+297, derived from this somatic hybrid as well as its BC₁ and BC₂ parental lines showed resistance to both tuber soft rot and early blight. Clone C75-5+297 has consistently out-yielded common varieties under disease stress. Using both molecular and cytogenetic approaches we demonstrated that a single copy of chromosome 8 from S. brevidens replaced a potato chromosome 8 in C75-5+297. Thus, C75-5+297 represents a potato-S. brevidens chromosome substitution line. Our results suggest that the presence of a single chromosome from S. brevidens may significantly impact the resistance to multiple potato diseases. The high yield potential of C75-5+297 makes it an excellent parent for developing potato varieties with resistances to both tuber soft rot and early blight.Communicated by J.S. Heslop-Harrison     

Will the Real Bemisia tabaci Please Stand Up?

Since Panayiotis Gennadius first identified the whitefly, Aleyrodes tabaci in 1889, there have been numerous revisions of the taxonomy of what has since become one of the world''s most damaging insect pests. Most of the taxonomic revisions have been based on synonymising different species under the name Bemisia tabaci. It is now considered that there is sufficient biological, behavioural and molecular genetic data to support its being a cryptic species complex composed of at least 34 morphologically indistinguishable species. The first step in revising the taxonomy of this complex involves matching the A. tabaci collected in 1889 to one of the members of the species complex using molecular genetic data. To do this we extracted and then amplified a 496 bp fragment from the 3′ end of the mitochondrial DNA cytochrome oxidase one (mtCOI) gene belonging to a single whitefly taken from Gennadius'' original 1889 collection. The sequence identity of this 123 year-old specimen enabled unambiguous assignment to a single haplotype known from 13 Mediterranean locations across Greece and Tunisia. This enabled us to unambiguously assign the Gennadius A. tabaci to the member of the B. tabaci cryptic species complex known as Mediterranean or as it is commonly, but erroneously referred to, as the ‘Q-biotype’. Mediterranean is therefore the real B. tabaci. This study demonstrates the importance of matching museum syntypes with known species to assist in the delimitation of cryptic species based on the organism''s biology and molecular genetic data. This study is the first step towards the reclassification of B. tabaci which is central to an improved understanding how best to manage this globally important agricultural and horticultural insect pest complex.