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131.
The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created through the insertion and deletion of specific uridylates. The editing reactions are required to be highly specific in order to ensure that functional open reading frames are created in edited mRNAs and that potentially deleterious modification of normally nonedited sequence does not occur. Selection-amplification and mutagenesis were previously used to identify the optimal sequence requirements for in vitro editing. There is, however, a minority of natural editing sites with suboptimal sequence. Several cis-acting elements, obtained from an in vitro selection, are described here that are able to compensate for a suboptimal editing site. An A + U sequence element within the 5'-untranslated region of cytochrome b mRNA from Leishmania tarentolae is also demonstrated to function as a cis-acting guide RNA and is postulated to compensate for a suboptimal editing site in vivo. Two proteins within an enriched editing extract are UV-cross-linked to two different in vitro selected editing substrates more efficiently than poorly edited RNAs. The results suggest that these proteins contribute to the specificity of the editing reaction.  相似文献   
132.
Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits. Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes. The release of bound RRF by EF-G is stimulated by GTP analogues. The EF-G-dependent release occurs in the presence of fusidic acid and viomycin. However, thiostrepton inhibits the release. RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF. On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex. In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity. These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA. Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF. We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly. Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site.  相似文献   
133.
Tracer methods for VLDL-TG kinetics vary in their ability to account for the effect of tracer recycling, which can influence the calculation of VLDL-TG fractional catabolic rates (FCRs). We evaluated a novel approach, involving stable isotopically labeled glycerol or palmitate tracers in conjunction with compartmental modeling, for measuring VLDL-TG kinetics in normolipidemic human subjects. When administered as a bolus simultaneously, both tracers provided identical VLDL-TG FCRs when the data were analyzed by a compartmental model that accounted for hepatic lipid tracer recycling, but not by non-compartmental analysis. The model-derived FCR was greater than that determined using a non-compartmental approach, and was 2- to 3-fold higher than that usually reported by using a bolus of radioactive [3H]glycerol. When palmitate tracer was given as a constant infusion, VLDL-TG turnover appeared 5-fold slower, because tracer recycling through hepatic lipid pools could not be resolved with the infusion protocol. We conclude that accounting for tracer recycling, particularly the contribution of hepatic glycerolipid pools, is essential to accurately measure VLDL-TG kinetics, and that bolus injection of stable isotopically labeled glycerol or palmitate tracers in conjunction with compartmental modeling analysis offers a reliable approach for measuring VLDL-TG kinetics.  相似文献   
134.
Tumor-directed therapeutic approaches require unique or overexpressed specific Ag or receptor as a target to achieve selective tumor killing. However, heterogeneous expression of these targets on tumor cells limits the efficacy of this form of therapy. In this study, we forced abundant expression of IL-13Ralpha2 chain by plasmid-mediated gene transfer in head and neck, as well as prostate tumors to provide a potential target. This was followed by successfully treating xenograft tumor-bearing nude mice with IL-13R-directed cytotoxin (IL13-PE38QQR). Although we did not observe an indirect cytotoxic bystander effect conveyed to nontransduced tumor cells in vitro, our approach in vivo led to a complete regression of established tumors transfected with IL-13Ralpha2 chain in most animals. We found that the tumor eradication was achieved in part by infiltration of macrophages and NK cells, assessed by immunohistochemistry. Moreover, head and neck tumors xenografted in macrophage-depleted nude mice were less sensitive to the antitumor effect of IL-13 cytotoxin. Because we did not observe vector-related toxicity in any vital organs, our novel combination strategy of gene transfer of IL-13Ralpha2 chain and receptor-directed cytotoxin therapy may be a useful approach for the treatment of localized cancer.  相似文献   
135.
C-reactive protein (CRP), the major human acute-phase plasma protein, binds to phosphocholine (PCh) residues present in pneumococcal C-polysaccharide (PnC) of Streptococcus pneumoniae and to PCh exposed on damaged and apoptotic cells. CRP also binds, in a PCh-inhibitable manner, to ligands that do not contain PCh, such as fibronectin (Fn). Crystallographic data on CRP-PCh complexes indicate that Phe(66) and Glu(81) contribute to the formation of the PCh binding site of CRP. We used site-directed mutagenesis to analyze the contribution of Phe(66) and Glu(81) to the binding of CRP to PCh, and to generate a CRP mutant that does not bind to PCh-containing ligands. Five CRP mutants, F66A, F66Y, E81A, E81K, and F66A/E81A, were constructed, expressed in COS cells, purified, and characterized for their binding to PnC, PCh-BSA, and Fn. Wild-type and F66Y CRP bound to PnC with similar avidities, while binding of E81A and E81K mutants to PnC was substantially reduced. The F66A and F66A/E81A mutants did not bind to PnC. Identical results were obtained with PCh-BSA. In contrast, all five CRP mutants bound to Fn as well as did wild-type CRP. We conclude that Phe(66) is the major determinant of CRP-PCh interaction and is critical for binding of CRP to PnC. The data also suggest that the binding sites for PCh and Fn on CRP are distinct. A CRP mutant incapable of binding to PCh provides a tool to assess PCh-inhibitable interactions of CRP with its other biologically significant ligands, and to further investigate the functions of CRP in host defense and inflammation.  相似文献   
136.
The use of the triterpenoid lupeol as a scaffold for the synthesis of lupeol-based libraries is described. Lupeol was anchored to a solid support (Rink amide/Sieber Amide) through aliphatic dicarboxylic acid moieties, which also served as a site for introducing diversity. The resulting polymer linked 3beta-O (resin-alkanoyl)-lup-20(29)-ene 3 was used to generate key intermediates 3beta-O (resin-alkanoyl)-30-bromo-lup-20(29)-ene 4 and 3beta-O (resin-alkanoyl)-30-amino-lup-20(29)-ene 6 for the generation of libraries based on disubstituted lupeol derivatives. A 96-member library was screened for its in-vitro antimalarial activity against Plasmodium falciparum.  相似文献   
137.
Through the development of a procedure to measure when hydrogen bonds form under two-state folding conditions, alpha-helices have been determined to form proportionally to denaturant-sensitive surface area buried in the transition state. Previous experiments assessing H/D isotope effects are applied to various model proteins, including lambda and Arc repressor variants, a coiled coil domain, cytochrome c, colicin immunity protein 7, proteins L and G, acylphosphatase, chymotrypsin inhibitor II and a Src SH3 domain. The change in free energy accompanied by backbone deuteration is highly correlated to secondary structure composition when hydrogen bonds are divided into two classes. The number of helical hydrogen bonds correlates with an average equilibrium isotope effect of 8.6 +/- 0.9 cal x mol(-1) x site(-1). However, beta-sheet and long-range hydrogen bonds have little isotope effect. The kinetic isotope effects support our hypothesis that, for helical proteins, hydrophobic association cannot be separated from helix formation in the transition state. Therefore, folding models that describe an incremental build-up of structure in which hydrophobic burial and hydrogen bond formation occur commensurately are more consistent with the data than are models that posit the extensive formation of one quantity before the other.  相似文献   
138.
Potent cytolytic peptides with specific tethering and cloaking sites have been synthesised and used to release payload from liposomes in a quantitative manner. A functionally located cloaking site has been modified specifically by simple conjugation without adversely affecting the cytolytic properties of the peptide. The cytolytic activity of modified peptides was then efficiently (>98%) cloaked and uncloaked by ligand-protein or hapten-antibody interactions. The principle of a dual response peptide has been demonstrated using an avidin-cloaked pH-sensitive peptide. Biospecific cloaking/uncloaking provided a new sensitive (approximately 12 pmol) homogeneous diagnostic and also appears potentially suited to bioresponsively targeted release of antimicrobial, anticancer and other drugs now delivered using liposomes.  相似文献   
139.
MOTIVATION: Arrays allow measurements of the expression levels of thousands of mRNAs to be made simultaneously. The resulting data sets are information rich but require extensive mining to enhance their usefulness. Information theoretic methods are capable of assessing similarities and dissimilarities between data distributions and may be suited to the analysis of gene expression experiments. The purpose of this study was to investigate information theoretic data mining approaches to discover temporal patterns of gene expression from array-derived gene expression data. RESULTS: The Kullback-Leibler divergence, an information-theoretic distance that measures the relative dissimilarity between two data distribution profiles, was used in conjunction with an unsupervised self-organizing map algorithm. Two published, array-derived gene expression data sets were analyzed. The patterns obtained with the KL clustering method were found to be superior to those obtained with the hierarchical clustering algorithm using the Pearson correlation distance measure. The biological significance of the results was also examined. AVAILABILITY: Software code is available by request from the authors. All programs were written in ANSI C and Matlab (Mathworks Inc., Natick, MA).  相似文献   
140.
Upreti RK  Kumar M  Shankar V 《Proteomics》2003,3(4):363-379
Although widely distributed in eukaryotic cells glycoproteins appear to be rare in prokaryotic organisms. The prevalence of the misconception that bacteria do not glycosylate their proteins has been a subject matter of discussion for a long time. Glycoconjugates that are linked to proteins or peptides, generated by the ribosomal translational mechanism have been reported only in the last two to three decades in a few prokaryotic organisms. Most studied prokaryotic glycoproteins are the S-layer glycoproteins of Archeabacteria. Apart from these, membrane-associated, surface-associated, secreted glycoproteins and exoenzymes glycoproteins are also well documented in both, Archea and Eubacteria. From the recent literature, it is now clear that prokaryotes are capable of glycosylating proteins. In general, prokaryotes are deprived of the cellular organelles required for glycosylation. In prokaryotes many different glycoprotein structures have been observed that display much more variation than that observed in eukaryotes. Besides following similar mechanisms in the process of glycosylation, prokaryotes have also been shown to use mechanisms that are different from those found in eukaryotes. The knowledge pertaining to the functional aspects of prokaryotic glycoproteins is rather scarce. This review summarizes developments and understanding relating to characteristics, synthesis, and functions of prokaryotic glycoproteins. An extensive summary of glycosylation that has been reported to occur in bacteria has also been tabulated. Various possible applications of these diverse biomolecules in biotechnology, vaccine development, pharmaceutics and diagnostics are also touched upon.  相似文献   
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