High yield regioselective monobenzyloxycarbonylation of secondary alcohols in glycopyranoside series

The regioselective monobenzyloxycarbonylation of secondary alcohols in methyl 6-O-(4-methoxytrityl)-alpha-D-manno-, gluco- and galactopyranoside has been achieved in high yields (74-85%) by using benzyl chloroformate in the presence of 4-dimethylaminopyridine and/or 1,4-diazabicyclo[2.2.2]octane.     

The first record of the swordtail Xiphophorus hellerii Heckel, 1848 (Poeciliidae,Actinopterygii) established in the wild from Morocco

Aquarium and ornamental fish trade is currently recognized as a major source of invasive species in aquatic ecosystems around the world. The swordtail Xyphophorus hellerii is a popular ornamental freshwater fish that occurs as an introduced species in the aquatic habitats of at least thirty-one countries. We report the first finding of an established population of the swordtail in Morocco. Twelve individuals of X. hellerii were captured along Oued Ain Chkef inside the park (Fez Province), including females and males (mature and immature). Ongoing investigations could soon reveal more occurrences of this species in the country.     

Development of a highly sensitive bacteria detection assay using fluorescent pH-responsive polymeric micelles

The detection and control of bacteria is extremely important in the safety of food products and health systems. The conventional microbiological methods based on culture enrichment techniques and plating procedures are highly sensitive and selective for bacterial detection but are expensive, cumbersome and time-consuming. Here we report the development of a simple and sensitive bioassay to detect Escherichia coli (E. coli) bacteria by using self assembled pH-responsive polymeric micelles that have been bioconjugated to anti-E. coli (capturing agent). Poly(ethylene glycol-b-trimethylsilyl methacrylate), containing silicon moieties that can be cleaved under mildly acidic conditions, was synthesized and self-assembled into micelles, that were loaded with a fluorescent dye (1-methylpyrene). The polymer silicon protecting groups are used as a tool to remotely activate the dye release by means of pH. The high sensitivity of the newly developed bioassay, which is capable of detecting 15 bacteria per milliliter of solution, is due to an amplification effect generated by the optical signal of millions of fluorophores released from a single micelle upon attachment to a bacterium. Fluorescence probing involves the measurements of changes in the emission spectra, through the disappearance of the excimer band, which only occurs when the dye molecules are trapped within the polymeric micelles.     

The Cytosolic Domain of Pex22p Stimulates the Pex4p-Dependent Ubiquitination of the PTS1-Receptor

Peroxisomal biogenesis is an ubiquitin-dependent process because the receptors required for the import of peroxisomal matrix proteins are controlled via their ubiquitination status. A key step is the monoubiquitination of the import receptor Pex5p by the ubiquitin-conjugating enzyme (E2) Pex4p. This monoubiquitination is supposed to take place after Pex5p has released the cargo into the peroxisomal matrix and primes Pex5p for the extraction from the membrane by the mechano-enzymes Pex1p/Pex6p. These two AAA-type ATPases export Pex5p back to the cytosol for further rounds of matrix protein import. Recently, it has been reported that the soluble Pex4p requires the interaction to its peroxisomal membrane-anchor Pex22p to display full activity. Here we demonstrate that the soluble C-terminal domain of Pex22p harbours its biological activity and that this activity is independent from its function as membrane-anchor of Pex4p. We show that Pex4p can be functionally fused to the trans-membrane segment of the membrane protein Pex3p, which is not directly involved in Pex5p-ubiquitination and matrix protein import. However, this Pex3(N)-Pex4p chimera can only complement the double-deletion strain pex4Δ/pex22Δ and ensure optimal Pex5p-ubiquitination when the C-terminal part of Pex22p is additionally expressed in the cell. Thus, while the membrane-bound portion Pex22(N)p is not required when Pex4p is fused to Pex3(N)p, the soluble Pex22(C)p is essential for peroxisomal biogenesis and efficient monoubiquitination of the import receptor Pex5p by the E3-ligase Pex12p in vivo and in vitro. The results merge into a picture of an ubiquitin-conjugating complex at the peroxisomal membrane consisting of three domains: the ubiquitin-conjugating domain (Pex4p), a membrane-anchor domain (Pex22(N)p) and an enhancing domain (Pex22(C)p), with the membrane-anchor domain being mutually exchangeable, while the Ubc- and enhancer-domains are essential.     

Versatile coordination behaviour of Ph2AsCH2AsPh2 with Ru(II)

Treatment of ‘RuCl₃ · 3H₂O’ with Ph₂AsCH₂AsPh₂ (dpam) in hot EtOH gives either trans-[RuCl₂(dpam-As,As′)(dpam-As)₂] (1), or cis-[RuCl₂(dpam-As,As′)₂] (2), depending on the mole ratio. On exposure to light, solutions of 2 isomerise to trans-[RuCl₂(dpam-As,As′)₂] (3). Treatment of [RuCl₂(PPh₃)₃] with two equivalents of dpam in CH₂Cl₂ gave a mixture of two products, from which trans-[RuCl₂(PPh₃) (dpam-As,As′)(dpam-As)] (4) was isolated by recrystallisation. The crystal structures of 1-4 are reported. Complexes 1-3 in CH₂Cl₂ undergo electrochemical oxidation to Ru(III), and the Ru(III) form of 2 undergoes isomerisation on the voltammetric timescale to the Ru(III) form of 3.     

The RING-type ubiquitin ligases Pex2p, Pex10p and Pex12p form a heteromeric complex that displays enhanced activity in an ubiquitin conjugating enzyme-selective manner

The RING finger peroxins Pex2p, Pex10p and Pex12p are central components of the peroxisomal matrix protein import machinery. The RING domain enables each of these proteins to exhibit ubiquitin-protein ligase activity, which has been linked to ubiquitin-dependent regulation of the peroxisomal import receptor Pex5p. The RING peroxins are considered to form a heteromeric complex in vivo, although the elucidation of the structural assembly, as well as the functional interplay of the RING domains, has remained elusive. Using in vitro approaches, we show that the RING domains form a heteromeric complex with Pex10p(RING) as a central component that directly binds the Pex2p(RING) and Pex12p(RING). The RING domains proved to function as heteromeric pairs that display an Pex10p-dependent enhanced ligase activity in an ubiquitin conjugating enzyme-selective manner.     

Distinct Ubiquitination Cascades Act on the Peroxisomal Targeting Signal Type 2 Co‐receptor Pex18p

Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by a peroxisomal targeting sequence (PTS) and ferry them to the peroxisomal membrane. Subsequently, the cargo is translocated into the peroxisomal lumen, whereas the receptor is released to the cytosol for further rounds of protein import. This cycle is controlled by the ubiquitination status of the receptor, which is best understood for the PTS1‐receptor. While polyubiquitination of PTS‐receptors results in their proteasomal degradation, the monoubiquitinated PTS‐receptors are exported to the cytosol and recycled for further rounds of protein import. Here, we describe the identification of two ubiquitination cascades acting on the PTS2 co‐receptor Pex18p. Using in vivo and in vitro approaches, we demonstrate that the polyubiquitination of Pex18p requires the ubiquitin‐conjugating enzyme (E2) Ubc4p, which cooperates with the RING (really interesting new gene)‐type ubiquitin‐protein ligases (E3) Pex2p as well as Pex10p. Monoubiquitination of Pex18p depends on the E2 enzyme Pex4p (Ubc10p), which functions in concert with the E3 enzymes Pex12p and Pex10p. Our findings for the PTS2‐pathway complement the data on PTS1‐receptor ubiquitination and add up to a unified concept of the ubiquitin‐based regulation of peroxisomal import .     

Cysteine-dependent ubiquitination of Pex18p is linked to cargo translocation across the peroxisomal membrane

The peroxisomal matrix protein import is facilitated by cycling receptor molecules that shuttle between the cytosol and the peroxisomal membrane. In the yeast Saccharomyces cerevisiae, the import of proteins harboring a peroxisomal targeting signal of type II (PTS2) is mediated by the receptor Pex7p and its co-receptor Pex18p. Here we demonstrate that Pex18p undergoes two kinds of ubiquitin modifications. One of these ubiquitination events depends on lysines 13 and 20 and forces rapid Pex18p turnover by proteasomal degradation. A cysteine residue near the extreme Pex18p amino-terminus is required for the second type of ubiquitination. It turned out that this cysteine residue at position 6 is essential for the function of Pex18p in peroxisomal protein import but does not contribute to receptor-cargo association and binding to the peroxisomal import apparatus. However, in contrast to the wild-type protein, cysteine 6-mutated Pex18p is arrested in a membrane-protected state, whereas Pex7p is accessible in a protease protection assay. This finding indicates that Pex18p export is linked to cargo translocation, which supports the idea of an export-driven import of proteins into peroxisomes.