The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.     

Cell mediated immune response elicited in mice after immunization with the P64k meningococcal protein: epitope mapping

The P64k protein of Neisseria meningitidis has been reported as an immunological carrier for weak immunogens. This investigation was aimed at characterizing the T-cell response produced in primed mice and at identifying T helper cell epitopes within this molecule. BALB/c mice subcutaneously immunized with the recombinant antigen provided inguinal lymph node cells (LNC) that proliferated in the presence of P64k in a dose-dependent manner. Proliferating cells secreted IL-4 while the concentration of IL-12 remained unaltered in the culture supernatant. By testing a panel of 59 overlapping synthetic peptides spanning the entire sequence of the antigen a T-cell determinant was localized. Prime-boost and lymphoproliferation experiments, conducted with highly purified synthetic peptides, confirmed that the segment including amino acids 470-485 comprises a T-cell epitope within the P64k molecule.     

Survival and growth of goat primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water

The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. We investigated the effectiveness of five culture media, two supplements and three culture periods on the survival and growth of goat primordial follicles after culturing ovarian cortex. The media were based on minimal essential minimum (MEM) and coconut water solution (CWS) added in the proportion of 0, 25, 50, 75 or 100%. The two supplements were none versus supplemented with insulin-transferrin-selenium, pyruvate, glutamine, hypoxanthine, and BSA. Pieces of goat ovarian cortex were cultured in the media for 1, 3 or 5 days and representative samples were evaluated at day 0 as non-cultured controls. The replicates were the two ovaries of five mixed breed goats. The number of primordial, intermediate, primary and secondary follicles at each period of culture and the number of degenerated follicles were evaluated. Mitotic activity of granulosa cells was studied by immunolocalization of proliferating cell nuclear antigen (PCNA). The number of follicles in each stage and degenerated follicles were statistically analyzed by ANOVA using a factorial design and the significance of differences assessed using Tukey test. Chi-square test was used to compare the percentage of follicles with PCNA positive granulosa cells. As the culture period progressed, the number of primordial follicles fell and there was a significant increase in the number of primary follicles. The fall in the number of primordial follicles was particularly marked after 1 day culture. No effect of media on the number of primordial and primary follicles was observed after culture, but MEM as well as supplements increased the number of intermediate follicles. Follicular degeneration was kept at the same level after culture in the media tested, except for pure CWS that increased the number of degenerated follicles. In contrast, addition of supplements to culture media reduced follicular degeneration. In non-cultured tissue, PCNA was expressed in granulosa cells of 31.6% of the growing follicles. This percentage had not significantly changed after 5 days culture in the various media, indicating the maintenance of proliferation activity of granulosa cells during culture. In conclusion, it is shown that goat primordial follicles may be successfully activated after in vitro culture in all media tested. However, when pure CWS is used the follicular degeneration is enhanced, but the addition of supplements to culture media decrease follicular degeneration.     

Steroidal saponins from the aerial parts of Tribulus pentandrus Forssk

Seven new steroidal glycosides named pentandrosides A(1)-G(7) were isolated from the EtOH extract of the aerial parts of Tribulus pentandrus. Pentandrosides A(1)-E(5) possess cholestane aglycones, pentandroside F(6) a furostan-type aglycone and pentandroside G(7) an unusual acyloxypregnane aglycone probably derived from the degradation of a furostan skeleton. Structure elucidation of 1-7 was accomplished through the extensive use of 1D- and 2D NMR experiments including 1H-1H (DQF-COSY, 1D-TOCSY) and 1H-13C (HSQC, HMBC) spectroscopy along with ESIMS and HRESIMS.     

Nitric oxide transfer from the NO-donor S-nitroso-N-acetylpenicillamine to monomers and dimers of water-soluble iron-porphyrins

The nitrosylation of two water-soluble iron-porphyrins, the anionic Fe(III)-meso-tetrakis(p-sulfonatophenyl)porphyrin (FeTPPS(4)) and the cationic Fe(III)-meso-tetrakis(4-N-methylpyridiniumyl)porphyrin (FeTMPyP), by the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) was studied using optical absorption spectroscopy. The influence of ionic and non-ionic micelles on rates of nitric oxide transfer was investigated. Initially, the effect of the micelles on the pH-dependent equilibrium between monomeric and micro-oxo-dimeric species of the iron-porphyrins was examined. It is not affected in micelle-porphyrin systems with electric charges identical in sign. The non-ionic micelles of polidocanol induce a small negative pK shift. In contrast, the dimerization equilibrium of porphyrins in oppositely charged micellar phases is displaced to lower pH by approximately 2 units. Nitric oxide binding to monomers and micro-oxo-dimers was examined at pH 5.0 and 8.0, respectively. Contrary to nitrosylation by NO gas, SNAP induces reductive nitrosylation. There was no observed NO-Fe(III)porphyrin intermediate. Nitrosylation rates were obtained and compared in aqueous buffer and in micellar systems. Monomers nitrosylate much faster than micro-oxo-dimers. Oppositely charged micelles prevent nitrosylation of the iron-porphyrins or considerably enhance nitrosylation times. Nitrosylation rates are comparable to transnitrosylation rates between several S-nitrosothiols and thiol-containing proteins, suggesting biological relevance for the process.     

Phenotypic characterization and description of two major O-serotypes in Tenacibaculum maritimum strains from marine fishes

Tenacibaculum maritimum is the etiological agent of marine flexibacteriosis disease, with the potential to cause severe mortalities in various cultured marine fishes. The development of effective preventive measures (i.e. vaccination) requires biochemical, serological and genetic knowledge of the pathogen. With this aim, the biochemical and antigenic characteristics of T. maritimum strains isolated from sole, turbot and gilthead sea bream were analysed. Rabbit antisera were prepared against sole and turbot strains to examine the antigenic relationships between the 29 isolates and 3 reference strains. The results of the slide agglutination test, dot-blot assay and immunoblotting of lipopolysaccharides (LPS) and membrane proteins were evaluated. All bacteria studied were biochemically identical to the T. maritimum reference strains. The slide agglutination assays using O-antigens revealed cross-reaction for all strains regardless of the host species and serum employed. However, when the dot-blot assays were performed, the existence of antigenic heterogeneity was demonstrated. This heterogeneity was supported by immunoblot analysis of the LPS, which clearly revealed 2 major serological groups that were distinguishable without the use of absorbed antiserum: Serotypes O1 and O2. These 2 serotypes seem to be host-specfic. In addition, 2 sole isolates and the Japanese reference strains displayed cross-reaction with both sera in all serological assays, and are considered to constitute a minor serotype, O1/O2. Analysis of total and outer membrane proteins revealed that all strains share a considerable number of common bands that are antigenically related.     

Synthesis and structure-activity relationships of 1,5-diazaanthraquinones as antitumour compounds

1,5-Diazaanthraquinone derivatives were synthesized employing single and double hetero Diels-Alder strategies. Their in vitro antitumour activity was assayed using three cell lines. Some of these compounds, specially those bearing methyl or ethyl groups at the C-3,7 positions or chloro at C-4 and methyl at C-7, showed IC(50) values in the 10(-8)M range for human lung carcinoma and human melanoma, which makes them attractive candidates for further development as anticancer agents.     

Fanconi anemia: contribution of molecular analyses to the identification of bone marrow graft donors and the study of chimerism in grafted patients

We report on the effectiveness of molecular studies regarding Fanconi anemia (FA) for a better selection of bone marrow graft donors and for post-transplant follow up. Ten unrelated FA patients and their families were analyzed by microsatellite markers. In 9 cases, the cytogenetic investigation of potential human leukocyte antigen (HLA)-identical related donors was normal, and the molecular analyses confirmed that they were also either normal or heterozygous carriers. For 1 patient, cytogenetic analysis of an HLA-identical sibling donor yielded ambiguous results with a relatively high number of chromosomal breakages using cross-linking agents. However, genotyping of this potential donor demonstrated his heterozygous state. Nine patients have received allogeneic bone marrow transplantation from HLA-matched related donors. Microsatellite analysis showed complete chimerism (CC) in all cases. The median follow up was 54 months (range 8-144 months). One patient out of 9 with CC rejected her graft without prior detection of a transitional mixed chimerism. Among these patients, 1 died 25 months after the transplantation of a chronic graft-versus-host-disease (GVHD). We conclude that, when the cytogenetic studies are not conclusive, molecular analyses are crucial to distinguish heterozygous carriers from asymptomatic FA Tunisian patients. Molecular analyses also allowed the evaluation of hematopoietic chimerism after allogeneic bone marrow transplantation and might be of value to identify patients with a high risk for graft rejection.     

Involvement of peptidorhamnomannan in the interaction of Pseudallescheria boydii and HEp2 cells

Pseudallescheria boydii is an emerging fungal pathogen that has a worldwide distribution. Virulence mechanisms of P. boydii are largely unknown. We studied the interaction between P. boydii and HEp2 cells and demonstrated that conidia of P. boydii attached to, and were ingested by, HEp2 cells in a time-dependent process. After 2 h of interaction, the conidia produced a germ-tube like projection, which was able to penetrate the epithelial cell membrane. Recently, our group characterized a peptidorhamnomannan (PRM) antigen on the cell surface of P. boydii. In order to better understand the role played by this surface glycoconjugate during cell adhesion and endocytosis, inhibition assays were performed using intact PRM and anti-PRM polyclonal antibody. When HEp2 cells were pre-treated with whole PRM molecule, the adhesion and endocytic indices were, respectively, 50% and 60% lower than in non-treated epithelial cells. Moreover, when the conidial cells were pre-incubated with anti-PRM antibodies, the adherence and endocytosis processes were inhibited in a dose-dependent manner. As PRM influenced the conidia P. boydii-HEp2 cell interaction, we also performed inhibition assays in order to observe which PRM moieties could be involved in this process. Treatment of PRM with proteinase K promoted a slight inhibition of adhesion. However, the de-O-glycosylated PRM molecule as well as the monosaccharide mannose was able to efficiently inhibit the adhesion and endocytic processes. In addition, our results indicate for the first time that P. boydii PRM binds to a polypeptide of 25 kDa on the HEp2 cell surface.