Characterization of the TRBP domain required for Dicer interaction and function in RNA interference

Background  

Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain.     

A regulatory light chain of ciliary outer arm dynein in Tetrahymena thermophila

Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed approximately 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.     

Quantitative assessment of methyl‐esterification and other side reactions in a standard propionylation protocol for detection of histone modifications

Histone modifications play an important role in regulating chromatin stability and gene expression, but to date, investigating them remains challenging. In order to obtain peptides suitable for MS‐based analysis, chemical derivatization of N‐terminus and lysine residues by propionic anhydride is commonly performed. Several side reactions (methyl‐esterification, amidation, solvolysis, overpropionylation, and missed propionylation) during propionylation protocols have been described, yet their relative abundances remain vague. Because methyl‐esterification could interfere with correct interpretation of the modification pattern, it is essential to take measures to avoid it. Here we present in‐depth quantitative analyses of methyl‐esterification and the other side reactions in a standard propionylation protocol containing methanol, and when replacing methanol with isopropanol or acetonitrile. We show that the use of alternative solvents can eliminate methyl‐esterification and that even though other side reactions are not prevented, their contribution can be kept relatively small. We also show that replacing methanol can be of importance also in other proteomics methods, such as mixed cation exchange, using methanol under acidic conditions.     

Self‐assembly of designed coiled coil peptides studied by small‐angle X‐ray scattering and analytical ultracentrifugation

α‐Helical coiled coil structures, which are noncovalently associated heptad repeat peptide sequences, are ubiquitous in nature. Similar amphipathic repeat sequences have also been found in helix‐containing proteins and have played a central role in de novo design of proteins. In addition, they are promising tools for the construction of nanomaterials. Small‐angle X‐ray scattering (SAXS) has emerged as a new biophysical technique for elucidation of protein topology. Here, we describe a systematic study of the self‐assembly of a small ensemble of coiled coil sequences using SAXS and analytical ultracentrifugation (AUC), which was correlated with molecular dynamics simulations. Our results show that even minor sequence changes have an effect on the folding topology and the self‐assembly and that these differences can be observed by a combination of AUC, SAXS, and circular dichroism spectroscopy. A small difference in these methods was observed, as SAXS for one peptide and revealed the presence of a population of longer aggregates, which was not observed by AUC. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Beta 1, 3-glucan recognition protein from the mosquito, Armigeres subalbatus, is involved in the recognition of distinct types of bacteria in innate immune responses

The activation of an immune response to invading microorganisms generally requires recognition by pattern recognition receptors. Beta 1, 3-glucan recognition proteins (GRPs) have specific affinity for beta 1, 3-glucan, a component on the surface of fungi and bacteria. In this study, we show that GRP from Armigeres subalbatus mosquitoes (AsGRP) is able to bind different bacterial species, and that this binding varies from species to species and is independent of Gram type. AsGRP knockdown with double-stranded RNA increases the mortality of mosquitoes to those bacteria that strongly bind AsGRP, but not to bacteria that do not detectably bind AsGRP. This increase in susceptibility is partially evidenced by decreased melanization in Salmonella typhimurium. Furthermore, AsGRP expression is differentially affected by the presence of different species of bacteria. These results demonstrate that AsGRP is selective in its affinity to different bacteria and; therefore, plays a role in the antibacterial immune response of mosquitoes.     

Analysis of gene order data supports vertical inheritance of the leukotoxin operon and genome rearrangements in the 5' flanking region in genus <Emphasis Type="Italic">Mannheimia</Emphasis>

Background  

The Mannheimia subclades belong to the same bacterial genus, but have taken divergent paths toward their distinct lifestyles. For example, M. haemolytica + M. glucosida are potential pathogens of the respiratory tract in the mammalian suborder Ruminantia, whereas M. ruminalis, the supposed sister group, lives as a commensal in the ovine rumen. We have tested the hypothesis that vertical inheritance of the leukotoxin (lktCABD) operon has occurred from the last common ancestor of genus Mannheimia to any ancestor of the diverging subclades by exploring gene order data.     

Motor unit activation patterns during concentric wrist flexion in humans with different muscle fibre composition

Muscle activity was recorded from the flexor carpi radialis muscle during static and dynamic-concentric wrist flexion in six subjects, who had exhibited large differences in histochemically identified muscle fibre composition. Motor unit recruitment patterns were identified by sampling 310 motor units and counting firing rates in pulses per second (pps). During concentric wrist flexion at 30% of maximal exercise intensity the mean firing rate was 27 (SD 13) pps. This was around twice the value of 12 (SD 5) pps recorded during sustained static contraction at 30% of maximal voluntary contraction, despite a larger absolute force level during the static contraction. A similar pattern of higher firing rates during dynamic exercise was seen when concentric wrist flexion at 60% of maximal exercise intensity [30 (SD 14) pps] was compared with sustained static contraction at 60% of maximal voluntary contraction [19 (SD 8) pps]. The increase in dynamic exercise intensity was accomplished by recruitment of additional motor units rather than by increasing the firing rate as during static contractions. No difference in mean firing rates was found among subjects with different muscle fibre composition, who had previously exhibited marked differences in metabolic response during corresponding dynamic contractions. It was concluded that during submaximal dynamic contractions motor unit firing rate cannot be deduced from observations during static contractions and that muscle fibre composition may play a minor role. Accepted: 5 May 1998     

Etlingera kenyalang (Zhgiberaceae) — a new species from Sarawak, Borneo, with notes on its ecology and Iban ethnobotany

Etlingera kenyalang is described from southern Sarawak. Ecological notes are given including a possible pollinator and seed dispersers. The Iban ethnobotany of the species is discussed; the shoots and fruits are edible and the leaves are used ritually during the Gawai Kenyalang (Hornbill Festival).