This work describes the design, synthesis and operation of a (R,R)-(+)-hydrobenzoin-diacetic acid molecular walker attached to polyether tracks containing 2, 3 or 4 secondary alcohol footholds. A macrocycle-the walker attached to a 2-foothold track by two ester linkages-was regioselectively hydrolysed by lipase AS. The resultant seco¬-acid was recyclised using Yamaguchi chemistry, demonstrating a bipedal walker stepping off, and on to, a track. These conditions were extended to tracks containing 3 and 4 alcohol footholds and the design of a 3-foothold track that incorporated a pentaethylene glycol chain. Using an information type Brownian ratchet mechanism, ~90% of walkers stepped away from the starting position, and ~68% of walkers took 2 steps to a foothold 16 atoms away. Importantly only smaller or equal to4% of walkers were found to completely detach from the track per operation cycle.

Yes / Attempts have been made to treat nonsense-associated genetic disorders by chemical agents and hence an improved mechanistic insight into the decoding of readthrough signals is essential for the identification and characterisation of factors for the treatment of these disorders. To identify either novel compounds or genes that modulate translation readthrough, we have employed dual reporter-based high-throughput screens that use enzymatic and fluorescence activities and screened bio-active NINDS compounds (n = 1000) and siRNA (n = 288) libraries. Whilst siRNAs targeting kinases such as CSNK1G3 and NME3 negatively regulate readthrough, neither the bio-active NINDS compounds nor PTC124 promote readthrough. Of note, PTC124 has previously been shown to promote readthrough. Furthermore, the impacts of G418 on the components of eukaryotic selenocysteine incorporation machinery have also been investigated. The selenocysteine machinery decodes the stop codon UGA specifying selenocysteine in natural selenoprotein genes. We have found that the eukaryotic SelC gene promotes the selenocysteine insertion sequence (SECIS)-mediated readthrough but inhibits the readthrough activity induced by G418. We have previously reported that SECIS-mediated readthrough at UGA codons follows a non-processive mechanism. Here, we show that G418-mediated promotion of readthrough also occurs through a non-processive mechanism which competes with translation termination. Based on our observations, we suggest that proteins generated through a non-processive mechanism may be therapeutically beneficial for the resolution of nonsense-associated genetic disorders. / Fellowship (awarded to MTN) from the Department of Health via the NIHR Comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London, Heptagon Life Science Proof of Concept Fund (grant KCL24 to MTN), the Great Britain Sasakawa 22 Foundation (grant B70 to MTN), the Royal Society (grant 43049 to MTN) and the University of Bradford (grants 003200 and DH005 to MTN).

<p>Degradation of the mRNA 3'-end located poly(A) tail is an important step for mRNA decay in mammalian cells. Thus, to understand mRNA decay in detail, it is important to identify the catalytic activities involved in degrading poly(A). We identified and purified a 54-kDa polypeptide responsible for poly(A)-specific 3' exonuclease activity in calf thymus extracts. The 54-kDa polypeptide is a proteolytic fragment of the poly(A)-specific ribonuclease (PARN) 74-kDa polypeptide. PARN is a divalent metal ion dependent, poly(A)-specific, oligomeric, processive and cap interacting 3' exonuclease. An active deadenylation complex, consisting of the poly(A)-tailed RNA substrate and PARN, has been identified. The interaction with the 5'-end cap structure stimulates PARN activity and also amplifies the processivity of the deadenylation reaction. Furthermore, the cap binding site and the active site of PARN are separate from each other. To characterise the active site of PARN, we per-formed side-directed mutagenesis, Fe²⁺-mediated hydroxyl radical cleavage and metal ion switch experiments. We have demonstrated that the conserved acidic amino acid residues D28, E30, D292 and D382 of human PARN are essential for PARN activity and that these amino acid residues are directly involved in the co-ordination of at least two metal ions in the active site of PARN. Phosphorothioate modification on RNA substrates revealed that the pro-R oxygen atom of the scissile phosphate group interacts directly with the metal ion(s). Based on our studies, we propose a model for the action of PARN. Similarly to what has been observed for ribozymes, aminoglycoside antibiotics inhibit PARN activity, most likely by the displacement of catalytically important divalent metal ions. Among the aminoglycoside antibiotics tested, neomycin B is the most potent inhibitor. We speculate that inhibition of enzymes using similar catalytic mechanisms as PARN could be a reason for the toxic side effects caused by aminoglycoside antibiotics in clinical practice. </p>

Degradation of the mRNA 3'-end located poly(A) tail is an important step for mRNA decay in mammalian cells. Thus, to understand mRNA decay in detail, it is important to identify the catalytic activities involved in degrading poly(A). We identified and purified a 54-kDa polypeptide responsible for poly(A)-specific 3' exonuclease activity in calf thymus extracts. The 54-kDa polypeptide is a proteolytic fragment of the poly(A)-specific ribonuclease (PARN) 74-kDa polypeptide. PARN is a divalent metal ion dependent, poly(A)-specific, oligomeric, processive and cap interacting 3' exonuclease. An active deadenylation complex, consisting of the poly(A)-tailed RNA substrate and PARN, has been identified. The interaction with the 5'-end cap structure stimulates PARN activity and also amplifies the processivity of the deadenylation reaction. Furthermore, the cap binding site and the active site of PARN are separate from each other. To characterise the active site of PARN, we per-formed side-directed mutagenesis, Fe2+-mediated hydroxyl radical cleavage and metal ion switch experiments. We have demonstrated that the conserved acidic amino acid residues D28, E30, D292 and D382 of human PARN are essential for PARN activity and that these amino acid residues are directly involved in the co-ordination of at least two metal ions in the active site of PARN. Phosphorothioate modification on RNA substrates revealed that the pro-R oxygen atom of the scissile phosphate group interacts directly with the metal ion(s). Based on our studies, we propose a model for the action of PARN. Similarly to what has been observed for ribozymes, aminoglycoside antibiotics inhibit PARN activity, most likely by the displacement of catalytically important divalent metal ions. Among the aminoglycoside antibiotics tested, neomycin B is the most potent inhibitor. We speculate that inhibition of enzymes using similar catalytic mechanisms as PARN could be a reason for the toxic side effects caused by aminoglycoside antibiotics in clinical practice.

Using English and the West-African creole language Krio as the objects of investigation, this study proposes an analysis in which verbs and the paradigms pertaining to verbs are conceived of as being the only direct carriers of linguistic time encoding. The fundamental assumption is that nominals encode substance, be it concrete or abstract, and that verbals encode abstract substance with time.The theoretical backdrop is provided by Derek Bickerton's Roots of Language (1981) and "The Language Bioprogram Hypothesis" (1984) in which he proposes a set of conceptually fundamental distinctions. These distinctions: the state/process; the durative/punctual; the realis/irrealis; and the anterior/non-anterior; are discussed in relation to four dynamicity values of verbal nuclei: stative; processive; eventive; and telic. These are proposed by the present author, but draw on Bernard Comrie's aspectual analysis in Aspect (1976).Three different layers of analysis are put forward: (1) the nucleic, which consists of the verbal carrying the meaning core of a situation; (2) the verbal constituency, in which we find all TMA encoding, that is, the tense, mood and aspect of the situation; and (3) the (verbal) situation, which is conceived of as a superordinate, maximum unit of description.It is argued that the dynamicity value of the verbal nucleus to a large extent determines and limits the possible aspectual, modal and temporal interpretations of the situation. / digitalisering@umu