FEBS Letters
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Pyrrolysine analogues as substrates for pyrrolysyl‐tRNA synthetase In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in‐frame UAG codons in the mRNAs of some methyltransferases. Pyl is directly acylated onto a suppressor tRNAPyl by pyrrolysyl‐tRNA synthetase (PylRS). Due to the lack of a readily available Pyl source, we looked for structural analogues that could be aminoacylated by PylRS onto tRNAPyl . We report here the in vitro aminoacylation of tRNAPyl by PylRS with two Pyl analogues: N‐ε‐d ‐prolyl‐l ‐lysine (d ‐prolyl‐lysine) and N‐ε‐cyclopentyloxycarbonyl‐l ‐lysine (Cyc). Escherichia coli , transformed with the tRNAPyl and PylRS genes, suppressed a lacZ amber mutant dependent on the presence of d ‐prolyl‐lysine or Cyc in the medium, implying that the E. coli translation machinery is able to use Cyc‐tRNAPyl and d ‐prolyl‐lysine‐tRNAPyl as substrates during protein synthesis. Furthermore, the formation of active β‐galactosidase shows that a specialized mRNA motif is not essential for stop‐codon recoding, unlike for selenocysteine incorporation.
FEBS Letters - Tập 580 Số 28-29 - Trang 6695-6700 - 2006
Approaches for chemically synthesized siRNA and vector‐mediated RNAi Successful applications of RNAi in mammalian cells depend upon effective knockdown of targeted transcripts and efficient intracellular delivery of either preformed si/shRNAs or vector expressed si/shRNAs. We have previously demonstrated that 27 base pair double stranded RNAs which are substrates for Dicer can be up to 100 times more potent than 21mer siRNAs. In this mini‐review we elaborate upon the rationale and design strategies for creating Dicer substrate RNAs that provide enhanced knockdown of targeted RNAs and minimize the utilization of the sense strand as RNAi effectors. Expression of shRNAs or siRNAs in mammalian cells can be achieved via transcription from either Pol II or Pol III promoters. There are certain constrictions in designing such vectors, and these are described here. Additionally, we review strategies for inducible shRNA expression and the various viral vectors that can be used to transduce shRNA genes into a variety of cells and tissues. The overall goal of this mini‐review is to provide an overview of available approaches for optimizing RNAi mediated down regulation of gene expression in mammalian cells via RNA interference. Although the primary focus is the use of RNAi mediated cleavage of targeted transcripts, it is highly probable that some of the approaches described herein will be applicable to RNAi mediated inhibition of translation and transcriptional gene silencing.
FEBS Letters - Tập 579 Số 26 - Trang 5974-5981 - 2005
The GroEL/GroES <i>cis</i> cavity as a passive anti‐aggregation device The GroEL/GroES chaperonin folding chamber is an encapsulated space of ∼65 Å diameter with a hydrophilic wall, inside of which many cellular proteins reach the native state. The question of whether the cavity wall actively directs folding reactions or is playing a passive role has been open. We review past and recent observations and conclude that the chamber functions as a passive “Anfinsen cage” that prevents folding monomers from multimolecular aggregation.
FEBS Letters - Tập 583 Số 16 - Trang 2654-2662 - 2009
The interaction between phenylalanine rings in proteins An analysis has been made of the geometry of phenylalanine‐phenylalanine interactions in proteins of known structure. 162 Phe‐Phe interactions were found with C‐C distances less than 4.6 Å. Three angles were used to define the geometry of interaction, P = the angle betwen ring planes, and polar coordinates, T θ, T ϕ to specify the relative spatial disposition of the two rings. The overall distribution of P values is the same as that expected for a random distribution of planes in 3 dimensions; i.e. the majority, of interactions have P approaching 90°. However, for high T θ values (when one Phe lies directly above the ring of the other Phe) the distribution is non‐random, and a preference for perpendicular interactions is expressed. This preference is in accord with recent quantum‐mechanical calculations.
FEBS Letters - Tập 191 Số 1 - Trang 1-6 - 1985
Effect of a negative regulatory element (NRE) on the human CYP1A1 gene expression in breast carcinoma MCF-7 and hepatoma HepG2 cells
FEBS Letters - Tập 365 - Trang 101-107 - 1995
Cysteine‐153 is required for redox regulation of pea chloroplast fructose‐1,6‐bisphosphatase Chloroplastic fructose‐1,6‐bisphosphatases are redox regulatory enzymes which are activated by the ferredoxin thioredoxin system via the reduction/isomerization of a critical disulfide bridge. All chloroplastic sequences contain seven cysteine residues, four of which are located in, or close to, an amino acid insertion region of approximately 17 amino acids. In order to gain more information on the nature of the regulatory site, five cysteine residues (Cys49 , Cys153 , Cys173 , Cys178 and Cys190 ) have been modified individually into serine residues by site‐directed mutagenesis. While mutations C173S and C178S strongly affected the redox regulatory properties of the enzyme, the most striking effect was observed with the C153S mutant which became permanently active and redox independent. On the other hand, the C190S mutant retained most of the properties of the wild‐type enzyme (except that it could now also be partially activated by the NADPH/NTR/thioredoxin h system). Finally, the C49S mutant is essentially identical to the wild‐type enzyme. These results are discussed in the light of recent crystallographic data obtained on spinach FBPase [Villeret et al. (1995) Biochemistry 34, 4299–4306].
FEBS Letters - Tập 401 Số 2-3 - Trang 143-147 - 1997
Characterization of thioredoxin <i>y</i>, a new type of thioredoxin identified in the genome of <i>Chlamydomonas reinhardtii</i> The sequencing of the Arabidopsis genome revealed a multiplicity of thioredoxins (TRX), ubiquitous protein disulfide oxido‐reductases. We have analyzed the TRX family in the genome of the unicellular green alga Chlamydomonas reinhardtii and identified eight different thioredoxins for which we have cloned and sequenced the corresponding cDNAs. One of these TRXs represents a new type that we named TRX y . This most probably chloroplastic TRX is highly conserved in photosynthetic organisms. The biochemical characterization of the recombinant protein shows that it exhibits a thermal stability profile and specificity toward target enzymes completely different from those of TRXs characterized so far.
FEBS Letters - Tập 543 - Trang 87-92 - 2003
Both oxidative and nitrosative stress are associated with muscle wasting in tumour‐bearing rats Reactive oxygen and nitrogen species (ROS and RNS) have been proposed as mechanisms of cancer‐induced cachexia. In this study, we assessed using Western blot analysis the levels of total protein carbonylation (2,4‐dinitrophenylhydrazine assay), both malondialdehyde‐ (MDA‐) and 2‐hydroxy‐4‐nonenal‐ (HNE‐) protein adducts, Mn‐superoxide dismutase (Mn‐SOD), catalase, heme oxygenase‐1 (HO‐1) and 3‐nitrotyrosine formation in gastrocnemius muscles of rats bearing the Yoshida AH‐130 hepatoma. In the muscles of the tumour‐bearing animals, protein carbonylation as measured by total levels of carbonyl group formation and both HNE and MDA‐protein adducts, and protein tyrosine nitration were significantly greater than in control muscles. Protein levels of the antioxidant enzymes Mn‐SOD, catalase, and HO‐1 were not significantly modified in the rat cachectic muscles compared to controls. The inefficiency of the antioxidant enzymes in neutralizing excessive ROS production may account for elevated markers of protein oxidation and be responsible for the development of both oxidative and nitrosative stress in cancer‐induced cachexia.
FEBS Letters - Tập 579 - Trang 1646-1652 - 2005
Purification of <i>N</i>‐acetyl β‐D‐hexosaminidase from bull epididymis by affinity chromatography
FEBS Letters - Tập 50 Số 1 - Trang 66-69 - 1975
Pertussis toxin‐dependent and ‐independent hormonal effects on cultured renal epithelioid cells The present study has been performed to test for the involvement of pertussis toxin‐sensitive GTP‐binding proteins (G‐proteins) in the cellular transduction of hormone‐induced activation of potassium channels. In Madin Darby canine kidney (MDCK) cells, a permanent cell line from dog kidney, epinephrine, acetylcholine, bradykinin, serotonin and ATP hyperpolarize the cell membrane by activation of potassium channels. In cells pretreated with pertussis toxin the hyperpolarizations elicited by either acetylcholine or serotonin are completely abolished; that following epinephrine is blunted and only transient. The hyperpolarizing effects of ATP or bradykinin are not affected by pertussis toxin. Thus, in MDCK cells both pertussis toxin‐dependent and ‐independent mechanisms operate in parallel to enhance the potassium conductance of the cell membrane.
FEBS Letters - Tập 234 Số 2 - Trang 263-266 - 1988
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