The ribosome as a molecular machine: the mechanism of tRNA-mRNA movement in translocation

Translocation of tRNA and mRNA through the ribosome is one of the most dynamic events during protein synthesis. In the cell, translocation is catalysed by EF-G (elongation factor G) and driven by GTP hydrolysis. Major unresolved questions are: how the movement is induced and what the moving parts of the ribosome are. Recent progress in time-resolved cryoelectron microscopy revealed trajectories of tRNA movement through the ribosome. Driven by thermal fluctuations, the ribosome spontaneously samples a large number of conformational states. The spontaneous movement of tRNAs through the ribosome is loosely coupled to the motions within the ribosome. EF-G stabilizes conformational states prone to translocation and promotes a conformational rearrangement of the ribosome (unlocking) that accelerates the rate-limiting step of translocation: the movement of the tRNA anticodons on the small ribosomal subunit. EF-G acts as a Brownian ratchet providing directional bias for movement at the cost of GTP hydrolysis.     

Expression,purification, and biochemical characterization of Chk,a soluble protein tyrosine kinase

CSK family contains two protein tyrosine kinases: Csk (C-terminal Src kinase) and Chk (Csk homologous kinase). They are responsible for phosphorylating Src family protein tyrosine kinases on a C-terminal Tyr (Tyr527) and negatively regulating their activities. However, Chk and Csk have different expression patterns, mechanisms of regulation, and different biological functions, and appear to play different roles in the development of breast cancer. To obtain pure human Chk for biochemical characterization, its coding region was amplified by polymerase chain reaction and expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The enzyme was highly expressed but unusually prone to proteolytic degradation during purification. Expression of the enzyme as a dual fusion protein with glutathione S-transferase on N-terminus and streptag, a 10 amino acid peptide, on C-terminus allowed purification of the full-length fusion protein. The purified enzyme was able to phosphorylate and inactivate Src. Chk (no inhibition up to 18.5 microM) and Csk (IC(50)= 1 microM) were differentially inhibited by PP2, probably due to the size difference of one residue (Thr265 in Csk versus Met304 in Chk) in the ATP-binding domain. The expression, purification, and initial characterizations of Chk provided an important step toward full characterization of Chk and Csk, two important enzymes in cellular regulation.     

Neuronal Ca2+ signaling via caldendrin and calneurons

The calcium sensor protein caldendrin is abundantly expressed in neurons and is thought to play an important role in different aspects of synapto-dendritic Ca2+ signaling. Caldendrin is highly abundant in the postsynaptic density of a subset of excitatory synapses in brain and its distinct localization raises several decisive questions about its function. Previous work suggests that caldendrin is tightly associated with Ca2+ - and Ca2+ release channels and might be involved in different aspects of the organization of the postsynaptic scaffold as well as with synapse-to-nucleus communication. In this report we introduce two new EF-hand calcium sensor proteins termed calneurons that apart from calmodulin represent the closest homologues of caldendrin in brain. Calneurons have a different EF-hand organization than other calcium sensor proteins, are prominently expressed in neurons and will presumably bind Ca2+ with higher affinity than caldendrin. Despite some significant structural differences it is conceivable that they are involved in similar Ca2+ regulated processes like caldendrin and neuronal calcium sensor proteins.     

Novel silicon-containing polyurethanes from vegetable oils as renewable resources. Synthesis and properties

Hydrosilylation of methyl 10-undecenoate (UDM) with phenyl tris(dimethylsiloxy)silane (PTDS) followed by a reduction of carboxylate groups was used to obtain a silicon-containing polyol with terminal primary hydroxyl groups (PSi194). Biobased silicon-containing polyurethanes, with a silicon content between 1.7% and 9.0%, were prepared from epoxidized methyl oleate-based polyether polyol (P184), PSi194, and 4,4'-methylenebis(phenyl isocyanate) (MDI). The thermal, mechanical, and flame-retardant properties of these materials were examined. The most notable change resulting from the incorporation of PSi194 is the appearance of melting endotherms of variable enthalpy and position and a downward shift in the T(g). The incorporation of silicon does not change the thermal stability but enhances the stability of the char under air atmosphere. Polyurethanes with higher silicon content no longer burn in ambient air without complementary oxygen, which suggests that these biobased materials are very interesting for applications that require fire resistance.     

Pulmonary aspergillosis outbreak in Rhea americana in Southern Brazil

Commercial raising of rheas is currently in expansion in the south of Brazil, and many diseases previously restricted to other avian species are currently emerging on rhea farms, especially as a result of careless management of these animals. The objective of the present article is to report a pulmonary aspergillosis outbreak that occurred in great rhea (Rhea americana) in the south of Brazil. About 50 birds aged 30 to 60 days died suddenly and one of them was submitted to autopsy which revealed the presence of white caseous nodules 0.5 mm in diameter occupying 95% of the lung area. One lung was sent to the Federal University of Santa Maria for histopathological and mycological analyses. Histopathological analysis revealed multifocal areas with necrosis and inflammatory infiltrates and the presence of fungal hyphae, giant cells and fibrous tissue proliferation at the periphery. Aspergillus fumigatus was recovered as pure culture from all culture media. This appears to be the first report of aspergillosis among great rhea in Brazil and the second in the world.     

Mice lacking JunB are osteopenic due to cell-autonomous osteoblast and osteoclast defects

Because JunB is an essential gene for placentation, it was conditionally deleted in the embryo proper. JunBDelta/Delta mice are born viable, but develop severe low turnover osteopenia caused by apparent cell-autonomous osteoblast and osteoclast defects before a chronic myeloid leukemia-like disease. Although JunB was reported to be a negative regulator of cell proliferation, junBDelta/Delta osteoclast precursors and osteoblasts show reduced proliferation along with a differentiation defect in vivo and in vitro. Mutant osteoblasts express elevated p16(INK4a) levels, but exhibit decreased cyclin D1 and cyclin A expression. Runx2 is transiently increased during osteoblast differentiation in vitro, whereas mature osteoblast markers such as osteocalcin and bone sialoprotein are strongly reduced. To support a cell-autonomous function of JunB in osteoclasts, junB was inactivated specifically in the macrophage-osteoclast lineage. Mutant mice develop an osteopetrosis-like phenotype with increased bone mass and reduced numbers of osteoclasts. Thus, these data reveal a novel function of JunB as a positive regulator controlling primarily osteoblast as well as osteoclast activity.