Elongation factor Tu can reduce translation errors in poly(U)-directed cell-free systems

Rates of incorporation of [³H]phenylalanine and [¹⁴C]leucine from the aminoacylated transfer-RNA into polypeptides synthesized on poly(U) programmed Escherichia coli ribosomes have been determined in cell-free translation systems containing either elongation factors Tu and G with GTP, or just elongation factor Tu or G with GTP, or none of the elongation factors. The presence of elongation factor Tu with GTP has been shown to reduce the leucine to phenylalanine ratio in the product at relatively low concentrations of Mg²⁺. This error-reducing effect of elongation factor Tu has not been observed at high concentrations of Mg²⁺, although the factor still contributed to the speed of elongation. The results are discussed in terms of the kinetic proof-reading mechanism proposed by Hopfield (1974).     

Formation and crystallization of Thermus thermophilus 70S ribosome/tRNA complexes

70S ribosomes from Thermus thermophilus are able to form ternary complexes with N-AcPhe-tRNA^Phe from either Thermus thermophilus or Escherichia coli, in the presence of a short oligo(U) of six or nine uridines. A complex of N-AcPhe-tRNA^Phe/(U)₉/70S ribosome from Th. thermophilus was crystallized under the same conditions used for the growth of crystals from isolated ribosomes (S.D. Trakhanov, et al., (1987) FEBS Lett. 220, 319–322).     

FoxO1在胰岛β细胞代谢灵活性受损及失代偿进程中的作用

葡萄糖及脂肪酸是胰岛β细胞的关键代谢底物,葡萄糖刺激胰岛β细胞分泌胰岛素是维持机体血糖稳态平衡的关键。胰岛素抵抗发生时,β细胞对能量代谢底物的选择失调,加速胰岛β细胞由代偿到胰岛β细胞失代偿的进程,是肥胖胰岛素抵抗最终发展为2型糖尿病的始动因素。核转录因子FoxO1属于Fox家族成员,在胰腺内广泛表达,在β细胞的代谢,发育,增殖过程中发挥着重要的调节作用。鉴于FoxO1在维持胰岛β细胞功能中的关键作用,现着重对FoxO1在胰岛β细胞代谢灵活性受损及失代偿过程发生中的作用调节进行阐述。为其作为调控胰岛β细胞功能的关键靶点提供参考。     

Separating the wheat from the chaff: unbiased filtering of background tandem mass spectra improves protein identification

Only a small fraction of spectra acquired in LC-MS/MS runs matches peptides from target proteins upon database searches. The remaining, operationally termed background, spectra originate from a variety of poorly controlled sources and affect the throughput and confidence of database searches. Here, we report an algorithm and its software implementation that rapidly removes background spectra, regardless of their precise origin. The method estimates the dissimilarity distance between screened MS/MS spectra and unannotated spectra from a partially redundant background library compiled from several control and blank runs. Filtering MS/MS queries enhanced the protein identification capacity when searches lacked spectrum to sequence matching specificity. In sequence-similarity searches it reduced by, on average, 30-fold the number of orphan hits, which were not explicitly related to background protein contaminants and required manual validation. Removing high quality background MS/MS spectra, while preserving in the data set the genuine spectra from target proteins, decreased the false positive rate of stringent database searches and improved the identification of low-abundance proteins.     

Modulation of activated oncogene c-kit expression with RNA-interference

Hyperexpression of oncogene c-kit is found in 80% patients with acute myeloid leukemia (AML). The transgenic model cell line expressing the oncogene c-kit was obtained by transduction with recombinant retrovirus. We have designed small interfering RNAs (siRNA) efficiently suppressing the expression of activated oncogene c-kit. Further small hairpin RNAs (shRNA) targeting c-kit mRNA were designed and expressed in lentiviral vectors. We report a stable reduction in c-kit expression following the introduction of shRNAs into model cells as well as Kasumi-1 cells from the patient with AML.