Visualization of the eEF2-80S ribosome transition-state complex by cryo-electron microscopy

In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-Å cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum tetrafluoride and GDP, with aluminum tetrafluoride mimicking the γ-phosphate during hydrolysis. This is the first visualization of a structure representing a transition-state complex on the ribosome. Tight interactions are observed between the factor's G domain and the large ribosomal subunit, as well as between domain IV and an intersubunit bridge. In contrast, some of the domains of eEF2 implicated in small subunit binding display a large degree of flexibility. Furthermore, we find support for a transition-state model conformation of the switch I region in this complex where the reoriented switch I region interacts with a conserved rRNA region of the 40S subunit formed by loops of the 18S RNA helices 8 and 14. This complex is structurally distinct from the eEF2-bound 80S ribosome complexes previously reported, and analysis of this map sheds light on the GTPase-coupled translocation mechanism.     

Effect of dietary supplements on lean mass and strength gains with resistance exercise: a meta-analysis.

The purpose of this study was to quantify which dietary supplements augment lean mass and strength gains during resistance training. Peer-reviewed studies between the years 1967 and 2001 were included in the analysis if they met a predetermined set of experimental criteria, among which were at least 3-wk duration and resistance-training 2 or more times a week. Lean mass and strength were normalized for meta-analysis by conversion to percent change per week and by calculating the effect size for each variable. Of the 250 supplements examined, only 6 had more than 2 studies that met the criteria for inclusion in the meta-analysis. Creatine and beta-hydroxy-beta-methylbutyrate (HMB) were found to significantly increase net lean mass gains of 0.36 and 0.28%/wk and strength gains of 1.09 and 1.40%/wk (P < 0.05), respectively. Chromium, dehydroepiandrosterone, androstenedione, and protein did not significantly affect lean gain or strength. In conclusion, two supplements, creatine and HMB, have data supporting their use to augment lean mass and strength gains with resistance training.     

Structural basis of the action of pulvomycin and GE2270 A on elongation factor Tu

Pulvomycin inhibits protein synthesis by preventing the formation of the ternary complex between elongation factor Tu (EF-Tu) x GTP and aa-tRNA. In this work, the crystal structure of Thermus thermophilus EF-Tu x pulvomycin in complex with the GTP analogue guanylyl imino diphosphate (GDPNP) at 1.4 A resolution reveals an antibiotic binding site extending from the domain 1-3 interface to domain 2, overlapping the domain 1-2-3 junction. Pulvomycin binding interferes with the binding of the 3'-aminoacyl group, the acceptor stem, and 5' end of tRNA. Only part of pulvomycin overlaps the binding site of GE2270 A, a domain 2-bound antibiotic of a structure unrelated to pulvomycin, which also hinders aa-tRNA binding. The structure of the T. thermophilus EF-Tu x GDPNP x GE2270 A complex at 1.6 A resolution shows that GE2270 A interferes with the binding of the 3'-aminoacyl group and part of the acceptor stem of aa-tRNA but not with the 5' end. Both compounds, pulvomycin more markedly, hinder the correct positioning of domain 1 over domains 2 and 3 that characterizes the active form of EF-Tu, while they affect the domain 1 switch regions that control the EF-Tu x GDP/GTP transitions in different ways. This work reveals how two antibiotics with different structures and binding modes can employ a similar mechanism of action.     

Uptake of benzyladenine in explants of Picea abies and Pinus sylvestris

Primary explants of Picea abies (L.) Karst. and Pinus sylvestris L. were incubated for various periods in solutions containing different concentrations of [¹⁴C]-benzyladenine. Time course analysis showed that uptake was linear for the initial 60 min; after this time linear uptake continued but at a much reduced rate. The amount of benzyladenine taken up by the explants saturated at a concentration about one third of that of the medium, Concentration dependence experiments showed that BA uptake was directly proportional to the external concentration. These results and those from experiments in which uptake was examined at different temperatures are consistent with a passive mode of BA uptake. The results are discussed with respect to in vitro micropropagation.     

High-resolution structure of a type IV pilin from the metal-reducing bacterium <Emphasis Type="Italic">Shewanella oneidensis</Emphasis>

Background

Type IV pili are widely expressed among Gram-negative bacteria, where they are involved in biofilm formation, serve in the transfer of DNA, motility and in the bacterial attachment to various surfaces. Type IV pili in Shewanella oneidensis are also supposed to play an important role in extracellular electron transfer by the attachment to sediments containing electron acceptors and potentially forming conductive nanowires.

Results

The potential nanowire type IV pilin Pil_Bac1 from S. oneidensis was characterized by a combination of complementary structural methods and the atomic structure was determined at a resolution of 1.67 Å by X-ray crystallography. Pil_Bac1 consists of one long N-terminal α-helix packed against four antiparallel β-strands, thus revealing the core fold of type IV pilins. In the crystal, Pil_Bac1 forms a parallel dimer with a sodium ion bound to one of the monomers. Interestingly, our Pil_Bac1 crystal structure reveals two unusual features compared to other type IVa pilins: an unusual position of the disulfide bridge and a straight α-helical section, which usually exhibits a pronounced kink. This straight helix leads to a distinct packing in a filament model of Pil_Bac1 based on an EM model of a Neisseria pilus.

Conclusions

In this study we have described the first structure of a pilin from Shewanella oneidensis. The structure possesses features of the common type IV pilin core, but also exhibits significant variations in the α-helical part and the D-region.

     

In-depth analysis of low abundant proteins in bovine colostrum using different fractionation techniques

Bovine colostrum is well known for its large content of bioactive components and its importance for neonatal survival. Unfortunately, the colostrum proteome is complicated by a wide dynamic range, because of a few dominating proteins that hamper sensitivity and proteome coverage achieved on low abundant proteins. Moreover, the composition of colostrum is complex and the proteins are located within different physical fractions that make up the colostrum. To gain a more exhaustive picture of the bovine colostrum proteome and gather information on protein location, we performed an extensive pre-analysis fractionation of colostrum prior to 2D-LC-MS/MS analysis. Physical and chemical properties of the proteins and colostrum were used alone or in combination for the separation of proteins. ELISA was used to quantify and verify the presence of proteins in colostrum. In total, 403 proteins were identified in the nonfractionated colostrum (NF) and seven fractions (F1-F7) using six different fractionation techniques. Fractionation contributed with 69 additional proteins in the fluid phase compared with NF. Different fractionation techniques each resulted in detection of unique subsets of proteins. Whey production by high-speed centrifugation contributed most to detection of low abundant proteins. Hence, prefractionation of colostrum prior to 2D-LC-MS/MS analysis expanded our knowledge on the presence and location of low abundant proteins in bovine colostrum.     

Splenocytes cultured in low concentrations of IL-2 generate NK cell specificities toward syngenic and allogenic targets

Splenocytes cultured in the presence of 30-60 units/ml IL-2 for 5 days develop natural killer activity toward syngeneic and allogeneic tumor cell targets. The IL-2 activated splenocytes, themselves, are partially resistant, whereas concanavalin A-activated T blast cells are completely resistant to killing. Surprisingly, major histocompatibility complex (MHC)-I-negative target cells are also resistant to natural killer (NK)-cell-mediated killing. Cells resistant to killing were unable to block NK-cell-mediated killing of sensitive targets as judged from cold target cell inhibition experiments, and one type of target cells sensitive to killing did generally not cross-block killing of other killing-sensitive target cell types. Alloantigen exposure of splenocytes, i.e., one-way mixed lymphocyte cultures, partially prevents the development of NK-cell activity. Our data suggest that target structures which trigger killing activity of NK cells are determined by the phenotype of the target cell and are dependent on its MHC class I expression disregarding the haplotype of the cell.