Nar1p, a conserved eukaryotic protein with similarity to Fe-only hydrogenases, functions in cytosolic iron-sulphur protein biogenesis.

The genome of the yeast Saccharomyces cerevisiae encodes the essential protein Nar1p that is conserved in virtually all eukaryotes and exhibits striking sequence similarity to bacterial iron-only hydrogenases. Previously, we have shown that Nar1p is an Fe-S protein and that assembly of its co-factors depends on the mitochondrial Fe-S cluster biosynthesis apparatus. Using functional studies in vivo, we demonstrated that Nar1p has an essential role in the maturation of cytosolic and nuclear, but not of mitochondrial, Fe-S proteins. Here we provide further spectroscopic evidence that Nar1p possesses two Fe-S clusters. We also show that Nar1p is required for Fe-S cluster assembly on the P-loop NTPase Nbp35p, another newly identified component of the cytosolic Fe-S protein assembly machinery. These data suggest a complex biochemical pathway of extra-mitochondrial Fe-S protein biogenesis involving unique eukaryotic proteins.

Molecular characterisation of aureocin A70, a multi-peptide bacteriocin isolated from Staphylococcus aureus.

Staphylococcus aureus A70 produces a heat-stable bacteriocin designated aureocin A70. Aureocin A70 is encoded within a mobilisable 8 kb plasmid, pRJ6, and is active against Listeria monocytogenes. Experiments of transposition mutagenesis and gene cloning had shown that aureocin A70 production and immunity were associated with the HindIII-A and B fragments of pRJ6. Therefore, a 6332 bp region of the plasmid, encompassing both these fragments, was sequenced using a concatenation DNA sequencing procedure. DNA sequence and genetic analyses revealed the presence of three transcriptional units that appear to be involved in bacteriocin activity. The first transcriptional unit contains a single gene, aurT, which encodes a protein that resembles an ATP-dependent transporter, similar to those involved in lantibiotic export. AurT is required for aureocin A70 production and it appears to be essential for mobilisation of pRJ6. The second putative operon contains two open reading frames (ORFs); the first gene, orfA, is predicted to encode a protein similar to small repressor proteins found in some Archaea, whose function remains to be elucidated. The second gene, orfB, codes for an 138 amino acid residue protein which shares a number of characteristics (high pI and hydrophobicity profile) with proteins associated with immunity, needed for self-protection against bacteriocin. Four other genes are present in the third operon, aurABCD. aurABCD encode four related peptides that are small (30-31 amino acid residues), strongly cationic (pI of 9.85 to 10.04) and highly hydrophobic. Theses peptides also have a high content of small amino acid residues like glycine and alanine, and no cysteine residue. Tn917-lac insertional mutations, which affected aureocin A70 activity, reside within operon aurABCD. Analysis of purified bacteriocin preparations by mass spectrometry demonstrated that all four peptides encoded by aurABCD operon are produced, expressed and excreted without post-translational modifications. Thus, aureocin A70 is a multi-peptide non-lantibiotic bacteriocin, which is transported without processing.

Potential use of gelcasting hydroxyapatite porous ceramic as an implantable drug delivery system.

Hydroxyapatite (HA) ceramic in a porous configuration is suggested as a drug release system. A new technique for the production of this material, based on the foaming of suspensions and in situ polymerization (gelcasting method), resulted in a material whose characteristics are likely to make it useful as an implantable drug delivery system. Three batches of HA ceramic with different porosities were characterized by X-ray diffraction and scanning electron microscopy (SEM). Pore size and shape as well as density were determined. In vitro experiments were performed in order to evaluate the dissolution behavior of cisplatin in the system. X-ray diffraction analysis showed that the final product consisted of a single phase, indicating that the sintering process had not affected the structure of the HA. Energy dispersive X-ray analysis (EDX) showed absence of impurities. Pore diameters were in the range 15--34 microm. SEM showed that the material presented a highly interconnected spheroidal porous network with open micropores and closed macropores. In vitro experiments showed significant differences in the release rate of cisplatin between three different porosities.