Efficient recovery of whole cell proteins in Oenococcus oeni—a comparison of different extraction protocols for high-throughput malolactic starter applications

In this study, we compared different total protein extraction protocols to achieve highly efficient isolation and purification of total proteins for the specific protein profiling of Oenococcus oeni. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns obtained for the different extraction protocols revealed not only a qualitative similar protein pattern but also quantitative variations with different intensity bands depending on the extraction method used. The selected extraction method added with sonication proved to work extremely well and efficiently and was able to obtain a high-resolution 2-D electrophoresis (2-DE) map. Prominent spots were successfully identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and corresponded to 76 different proteins involved in the main metabolic pathways. The approach allowed to achieve a protein profiling specific for O. oeni from Aglianico wine with numerous characterized protein products corresponding to many different O. oeni genes and associated with main cellular pathways. Further investigations of the 2-DE protein expression profile will provide useful and interesting information on the molecular mechanisms at the protein level responsible for growth and survival of O. oeni in wine.     

The use and limits of ITS data in the analysis of intraspecific variation in Passiflora L. (Passifloraceae)

The discovery and characterization of informative intraspecific genetic markers is fundamental for evolutionary and conservation genetics studies. Here, we used nuclear ribosomal ITS sequences to access intraspecific genetic diversity in 23 species of the genus Passiflora L. Some degree of variation was detected in 21 of these. The Passiflora and Decaloba (DC.) Rchb. subgenera showed significant differences in the sizes of the two ITS regions and in GC content, which can be related to reproductive characteristics of species in these subgenera. Furthermore, clear geographical patterns in the spatial distribution of sequence types were identified in six species. The results indicate that ITS may be a useful tool for the evaluation of intraspecific genetic variation in Passiflora.     

Chromosome phylogeny of the subfamily Pitheciinae (Platyrrhini,Primates) by classic cytogenetics and chromosome painting

Background  

The New World monkey (Platyrrhini) subfamily Pitheciinae is represented by the genera Pithecia, Chiropotes and Cacajao. In this work we studied the karyotypes of Pithecia irrorata (2n = 48) and Cacajao calvus rubicundus (2n = 45 in males and 2n = 46 in females) by G- and C-banding, NOR staining and chromosome painting using human and Saguinus oedipus whole chromosome probes. The karyotypes of both species were compared with each other and with Chiropotes utahicki (2n = 54) from the literature.     

Direct anti-metastatic efficacy by the DNA enzyme Dz13 and downregulated MMP-2, MMP-9 and MT1-MMP in tumours

The DNA enzyme Dz13, targeted against the oncogene c-Jun, is capable of inhibiting various model tumours in mice albeit in ectopic models of neoplasia. In previous studies using orthotopic models of disease, the inhibitory effects of Dz13 on secondary growth was a direct result of growth inhibition at the primary lesion site. Thus, the direct and genuine effects on metastasis were not gauged. In this study, Dz13 was able to inhibit both locoregional and distal metastasis of tumour cells in mice, in studies where the primary tumours were unaffected due to the late and clinically-mimicking nature of treatment commencement. In addition, the effect of Dz13 against tumours has now been extended to encompass breast and prostate cancer. Dz13 upregulated the matrix metalloproteinase (MMP)-2 and MMP-9, and decreased expression of MT1-MMP (MMP-14) in cultured tumour cells. However, in sections of ectopic tumours treated with Dz13, both MMP-2 and MMP-9 were downregulated. Thus, not only is Dz13 able to inhibit tumour growth at the primary site, but also able to decrease the ability of neoplastic cells to metastasise. These findings further highlight the growing potential of Dz13 as an antineoplastic agent.     

Molecular Phylogenetic Analysis of Petunia Juss. (Solanaceae)

Representatives from 11 Petunia Jussieu species in south and southeast Brazil were compared with two Calibrachoa La Llave & Lex., one Bouchetia Dunal, and two Nierembergia Ruiz & Pav. taxa in relation to DNA molecular variability. A total of 4532 base pairs related to one nuclear, five plastidial and one mitochondrial systems was investigated. Petunia and Calibrachoa, although separated among themselves, clearly differentiate from the two other genera. Despite the fact that the Petunia species do not show marked molecular differences, they can be separated in two complexes, in good agreement with altitude data. Petunia + Calibrachoa should have diverged from other clades at about 25 million years before present.     

Comparative proteomics and immunoproteomics of Helicobacter pylori related to different gastric pathologies

Helicobacter pylori is a Gram-negative bacterium which causes ulcer, atrophic gastritis, adenocarcinoma, or mucosa-associated lymphoid tissue lymphoma. A comparative proteomic and immunoproteomic analysis was chosen to identify the antigenic patterns of three different H. pylori strains. These strains were probed against single sera from H. pylori-positive patients affected by gastric adenocarcinoma or duodenal ulcer. We found a quite heterogeneous antigenic pattern, both from strain and sera points of view, thus underlying both a strain- and a host-specificity. The different antigenic repertoires introduced the importance of the strain to be used for immunoblotting as a diagnostic test.     

Biologically relevant effects of mRNA amplification on gene expression profiles

Background  

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results.     

Conjugal transfer of plasmid-borne bacteriocin production in Enterococcus faecalis 226 NWC

Enterococcus faecalis 226 NWC, isolated from natural whey cultures utilized as starter in water-buffalo Mozzarella cheese manufacture, produces a bacteriocin, designated Enterocin 226 NWC, which is inhibitory to Listeria monocytogenes. Plasmid analysis of E. faecalis 226 NWC showed a single 5.2-kb plasmid, pEF226. In conjugation experiments, pEF226 was transferred into a plasmid-free strain of E. faecalis JH2-2. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The identity of conjugation was confirmed by digestion with SmaI restriction endonuclease and subsequent pulsed-field gel electrophoresis (PFGE) of the genomic DNA of E. faecalis 226, E. faecalis JH2-2 and of the isolates after the mating. The data indicate that the ability of E. faecalis 226 NWC to produce the bacteriocin is linked to the 5.2-kb conjugative plasmid pEF226.     

Genetic and nongenetic influences on the ABH and Lea antigen levels of saliva and milk.

The saliva and milk of 250 parturient women were studied in relation to ABH antigen levels; part of the sample was also investigated for the Lewis (Lea) substance. The levels of A and B are higher in saliva, and those of H and Lea higher in milk. The H average salivary titers presented the relationship O greater than A2 greater than A1 greater than B greater than AB, but these differences were not present in milk. In addition, the salivary levels of A and B are similar in individuals of these groups but B greater than A in AB persons, and A1 greater than A2; while in milk A greater than B in A, B and AB subjects, and A1 approximately equal to A2. The amount of Lea substance depends of the ABH secretor status in both secretions; but independently of this difference, the average titers were always higher in milk. Correlation coefficients between the levels observed in the two secretions are statistically significant for the A substance in A persons (0.46), H in B (0.58) and Lea in all subjects tested (0.47). A stepwise multiple regression analysis performed to verify the influence of four genetic and six nongenetic variables in the ABH levels of both fluids indicated only one consistent modifying factor: ABO type.