The transporter CefM involved in translocation of biosynthetic intermediates is essential for cephalosporin production

The cluster of early cephalosporin biosynthesis genes (pcbAB, pcbC, cefD1, cefD2 and cefT of Acremonium chrysogenum) contains all of the genes required for the biosynthesis of the cephalosporin biosynthetic pathway intermediate penicillin N. Downstream of the cefD1 gene, there is an unassigned open reading frame named cefM encoding a protein of the MFS (major facilitator superfamily) with 12 transmembrane domains, different from the previously reported cefT. Targeted inactivation of cefM by gene replacement showed that it is essential for cephalosporin biosynthesis. The disrupted mutant accumulates a significant amount of penicillin N, is unable to synthesize deacetoxy-, deacetyl-cephalosporin C and cephalosporin C and shows impaired differentiation into arthrospores. Complementation of the disrupted mutant with the cefM gene restored the intracellular penicillin N concentration to normal levels and allowed synthesis and secretion of the cephalosporin intermediates and cephalosporin C. A fused cefM-gfp gene complemented the cefM-disrupted mutant, and the CefM-GFP (green fluorescent protein) fusion was targeted to intracellular microbodies that were abundant after 72 h of culture in the differentiating hyphae and in the arthrospore chains, coinciding with the phase of intense cephalosporin biosynthesis. Since the dual-component enzyme system CefD1-CefD2 that converts isopenicillin N into penicillin N contains peroxisomal targeting sequences, it is probable that the epimerization step takes place in the peroxisome matrix. The CefM protein seems to be involved in the translocation of penicillin N from the peroxisome (or peroxisome-like microbodies) lumen to the cytosol, where it is converted into cephalosporin C.     

Morphological and anatomical evaluation of adult and juvenile leaves of olive plants

The olive tree (Olea europaea L.), like many other woody plants, has a long juvenile period in which the plant is not able to produce flowers. Knowledge of the moment when the plant is capable of flowering is important for breeding programs and also for determining the physiological basis for sexual reproductive behavior, but currently the only indicator of that moment is the actual flowering. In many species, the juvenile-to-adult phase shift includes changes in leaf structure known as heteroblasty, that is, varied form of successive leaves on the same plant. Some differences have been observed between juvenile and adult olive leaves, particularly in size and form, but to our knowledge, no complete systematic study has been carried out. In this research, we measured size, morphology and anatomy for juvenile and adult leaves of olive plants grown from seeds. Differences were found in most of the parameters studied, including leaf size, form, mesophyll thickness, layers of palisade parenchyma and quantity of peltate trichomes, which were generally significant but overlapping between the two leaf types. The most consistent and striking difference was the presence of an organized layer of subepidermal cells only in the abaxial mesophyll of adult leaves. This characteristic could be a simple and effective criterion of phase change in the olive tree.     

Study on Cortinarius subgenus Telamonia section Hydrocybe in Europe, with especial emphasis on Mediterranean taxa

In this paper we have attempted to clarify the taxonomy and nomenclature of thirteen taxa of the genus Cortinarius subgenus Telamonia (sections Hydrocybe, Fraternii) well represented in the southwestern Mediterranean area of Europe (C. atrocoeruleus, C. bombycinus, C casimiri, C. contrarius, C. decipiens, C. fraternus, C. gallurae, C. hoffmannii, C. petroselineus, C. sertipes, C. subturibulosus, C. urdaibaiensis and C. vernus). To this end we have performed a combined study of morphological and molecular data (rDNA ITS sequences). The morphological analysis was carried out on 114 collections and the molecular analysis involved 31 of the 114 collections, including 11 type collections (types for C. casimiri and C. fraternus were not available). In addition, a study of spores under field emission scanning electron microscopy (FESEM) was conducted. The results of the combined analysis allowed us to asign the studied material to five species (C. casimiri s.l., C. decipiens s.l., C. gallurae, C. subturibulosus s.l. and C. vernus s.l.). Thus, all collections from more continental areas, which were originally identified as six different taxa (C. atrocoeruleus, C. contrarius, C. decipiens, C. fraternus, C. sertipes, C. flexipes fo. sertipes) corresponded to C. decipiens sensu lato, a widely distributed, genetically and morphologically variable species. Cortinarius casimiri is also found in such habitats, but it is confirmed as distinct taxon. Collections from Mediterranean sclerophyllous communities correspond to C. gallurae, C. vernus sensu lato and C. subturibulosus sensu lato. Due to close phylogenetic relationships we propose the new combinations C. casimiri var. hoffmannii (=C. decipiens var. hoffmannii non C. hoffmannii) and C. subturibulosus var. bombycinus (=C. bombycinus), and the new variety C. vernus var. nevadavernus (=C. vernus H. Lindstr. & Melot sensu auct.).     

Crystal structure of CbpF,a bifunctional choline‐binding protein and autolysis regulator from Streptococcus pneumoniae

Phosphorylcholine, a crucial component of the pneumococcal cell wall, is essential in bacterial physiology and in human pathogenesis because it binds to serum components of the immune system and acts as a docking station for the family of surface choline‐binding proteins. The three‐dimensional structure of choline‐binding protein F (CbpF), one of the most abundant proteins in the pneumococcal cell wall, has been solved in complex with choline. CbpF shows a new modular structure composed both of consensus and non‐consensus choline‐binding repeats, distributed along its length, which markedly alter its shape, charge distribution and binding ability, and organizing the protein into two well‐defined modules. The carboxy‐terminal module is involved in cell wall binding and the amino‐terminal module is crucial for inhibition of the autolytic LytC muramidase, providing a regulatory function for pneumococcal autolysis.     

Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum

Background  

Penicillium chrysogenum converts isopenicillin N (IPN) into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT), which is encoded by the penDE gene. In silico analysis of the P. chrysogenum genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding penDE gene. We have termed this gene ial because it encodes a protein with high similarity to IAT (IAL for IAT-Like). We have conducted an investigation to characterize the ial gene and to determine the role of the IAL protein in the penicillin biosynthetic pathway.     

Intronic hammerhead ribozymes are ultraconserved in the human genome

Small ribozymes have been regarded as living fossils of a prebiotic RNA world that would have remained in the genomes of modern organisms. In this study, we report the ultraconserved occurrence of hammerhead ribozymes in Amniota genomes (reptiles, birds and mammals, including humans), similar to those described previously in amphibians and platyhelminth parasites. The ribozymes mapped to intronic regions of different genes, such as the tumour suppressor RECK in birds and mammals, a mammalian tumour antigen and the dystrobrevin beta in lizards and birds. In vitro characterization confirmed a high self-cleavage activity, whereas analysis of RECK-expressed sequence tags revealed fusion events between the in vivo self-cleaved intron and U5 or U6 small nuclear RNA fragments. Together, these results suggest a conserved role for these ribozymes in messenger RNA biogenesis.     

N',2-diphenylquinoline-4-carbohydrazide based NK3 receptor antagonists

A new class of potent NK3R antagonists based on the N',2-diphenylquinoline-4-carbohydrazide core is described. In an ex vivo assay in gerbil, the lead compound 2g occupies receptors within the CNS following oral dosing (Occ(90) 30 mg/kg po; plasma Occ(90) 0.95 microM) and has good selectivity and promising PK properties.     

Cell Adhesion and Proliferation on Sulfonated and Non-Modified Chitosan Films

Three types of chitosan-based films have been prepared and evaluated: a non-modified chitosan film bearing cationizable aliphatic amines and two films made of N-sulfopropyl chitosan derivatives bearing both aliphatic amines and negative sulfonate groups at different ratios. Cell adhesion and proliferation on chitosan films of C2C12 pre-myoblastic cells and B16 cells as tumoral model have been tested. A differential cell behavior has been observed on chitosan films due to their different surface modification. B16 cells have shown lower vinculin expression when cultured on sulfonated chitosan films. This study shows how the interaction among cells and material surface can be modulated by physicochemical characteristics of the biomaterial surface, altering tumoral cell adhesion and proliferation processes.