In vitro production of cat blastocysts of predetermined sex using flow cytometrically sorted semen

Sex preselection in cats can have applications for both breeding purposes and as an experimental model for endangered felids. The present study examined the ability to produce cat embryos from in vitro fertilization (IVF) of in vitro matured (IVM) cat oocytes with flow cytometrically sorted spermatozoa and to verify the sex of the embryos obtained from sexed spermatozoa by PCR. In the first experiment, a total of 224 oocytes were fertilized with spermatozoa from six ejaculates sorted without sex separation. The sorting process did not influence the cleavage rate (sorted 44.0% versus unsorted 46.1%), day 6 morula-blastocyst rate (sorted 26.6% versus unsorted 29.6%) and day 7 blastocyst rate (sorted 16.5% versus unsorted 16.5%). In the second experiment, a total of 84 IVM oocytes were fertilized with sorted X- and Y-chromosome bearing spermatozoa from four ejaculates in order to obtain embryos of preselected sex. Embryonic sex determination by PCR revealed that 21 out of 24 embryos reaching morula/blastocyst stage (87.5%) were of the desired sex. In particular 12 out of 14 embryos (85.7%) derived from X-bearing spermatozoa were female and 9 embryos out of 10 (90%) derived from Y-bearing spermatozoa were male. Our results show, for the first time, that X- and Y-chromosome bearing spermatozoa sorted by high-speed flow cytometry can be successfully used in an IVM-IVF system to obtain cat embryos of a predetermined sex.     

Wounding induces resistance to pathogens with different lifestyles in tomato: role of ethylene in cross-protection

Many reports point to the existence of a network of regulatory signalling occurring in plants during the interaction with micro-organisms (biotic stress) and abiotic stresses such as wounding. However, the focus is on shared intermediates/components and/or common molecular outputs in differently triggered signalling pathways, and not on the degree and modes of effective influence between abiotic and biotic stresses nor the range of true plant-pathogen interactions open to such influence. We report on local and systemic wound-induced protection in tomato (Solanum lycopersicum L.) to four pathogens with a range of lifestyles (Botrytis cinerea, Fusarium oxysporum f.sp. lycopersici, Phytophthora capsici and Pseudomonas syringae pv. tomato). The role of ethylene (ET) in the phenomenon and in the induction by wounding of several markers of defense was investigated by using the never-ripe tomato mutant plants impaired in ET perception. We showed that PINIIb, PR1b, PR5, PR7 and peroxidase (POD) are influenced locally and/or systemically by wounding and, with the exception of POD activity, by ET perception. We also demonstrated that ET, although not essential, is positively (B. cinerea, P. capsici) or negatively (F. oxysporum, P. syringae pv. tomato) involved not only in basal but also in wound-induced resistance to each pathogen.     

Typology of environmental conditions at the onset of winter phytoplankton blooms in a shallow macrotidal coastal ecosystem, Arcachon Bay (France)

Phytoplankton dynamics were assessed in the macrotidal ecosystemof Arcachon Bay through high-frequency surveys over a 5-yearperiod in order to characterize typology of environmental conditionsat the onset of the productive period. Temporal variations ofhydrological and biological parameters were examined in externaland internal waters of the lagoon, during the winter–springperiods from 1999 to 2003. An additional survey was performedduring winter–spring 2005 in order to study the verticalstructure of the water column. The occurrence of winter phytoplanktonblooms between January and March emerged as a recurrent event.The early onset of the productive period is influenced by thebiological functioning of adjacent Bay of Biscay oceanic waters.It is hypothesized that under a propitious hydrodynamic regime,phytoplankton inocula from the Bay of Biscay enter in the ArcachonBay where cells presumably find favourable conditions for theirfast development. The timing of the onset of those winter bloomsin Arcachon Bay seems to be mainly influenced by the presenceof anticyclonic weather conditions (associated with an increasein incident irradiance) during late winter (i.e. by February),while the water column does not show any particular stabilizationnor stratification liable to facilitate the onset of these blooms.Moreover, these winter blooms dominated by diatoms led to anearly nutrient depletion which could have inevitable consequenceson the structuration of the food web during spring and summer.     

Occurrence and diversity of Frankia in Tunisian soil

There is a lack of studies on the occurrence and diversity of Frankia in African soils, including those in northern African regions. The present study on Tunisian soils is an attempt to address this issue using Alnus glutinosa , Elaeagnus angustifolia and Casuarina glauca in a plant capturing bioassay on 30 soil samples, followed by amplified 16S ribosomal DNA restriction pattern analysis (ARDRA). A total of seven ARDRA haplotypes of Frankia have been detected in root actinorhizas that have been affiliated to theoretical ARDRA haplotypes upon in silico digestion of selected 16S ribosomal RNA (rRNA) gene sequences retrieved from GeneBank and confirmed by their partial 16S rRNA gene sequencing. Elaeagnus -compatible Frankia isolates were widespread and form four ARDRA haplotypes affiliated to Frankia , colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic subgroups. Alnus -compatible strains occurring in northern subhumid area were closely related to Alnus – Morella -compatible strains and clustered in two ARDRA haplotypes. Casuarina -compatible strains lack variability in several northern arboreta. The relatively wide diversity of Tunisian Frankia strains opens the perspective that African soil could be an interesting reservoir for the isolation of new actinorhizal strains that could be used as potential biofertilizers to counteract the progressive soil desertification which indeed is a crucial environmental problem in Northern Africa.     

Glutathione S-transferases in Festuca arundinacea: identification, characterization and inducibility by safener benoxacor

Over recent years it has emerged how certain no crop-species can be employed in phytoremediating contaminated soils or preventing herbicide pollution; in this contest Festuca arundinacea was investigated. Shoots of Festuca were submitted to fast protein liquid chromatography in order to identify their glutathione S-transferases (GST; EC 2.5.1.18), by a combination of anionic, affinity and RP-HPLC chromatography. The chromatographic procedure revealed satisfactory yield and four GSTs were identified: they were named FaGST I, FaGST II, FaGST III and FaGST IV. Among these, significant differences were observed in the chromatographic behaviours, structure, activity toward a "model" substrate, 1-chloro-2,4-dinitrobenzene, and responsiveness to the herbicide safener benoxacor. FaGST I showed the highest activity toward the above substrate, and this activity was up-regulated by the herbicide safener. Therefore, FaGST I was purified till homogeneity and was determined to be an heterodimer consisting of two subunits of 28.0 and 27.2kDa. Each subunit of FaGST I was further characterized by means of LC-ESI-MS/MS and immunoblotting analysis, which revealed that both the subunits belong to the tau subclass.     

Isolation and characterization of heterotepalins, type 1 ribosome-inactivating proteins from Phytolacca heterotepala leaves

Leaves from Phytolacca heterotepala H. Walter (Mexican pokeweed) contain at least 10 type 1 RIP isoforms, named heterotepalins. Their Mr values are included in the range 28,000-36,000, as shown by SDS-PAGE performed under reduced conditions and the pI values in the pH range 8.50-9.50. Some heterotepalins are glycosylated. ESI-QTOF mass spectrometry provides the accurate Mr of heterotepalin 4 (29,326.00) and heterotepalin 5b (30,477.00), two isoforms purified to homogeneity by conventional chromatographic techniques. The N-terminal sequences up to residue 35, show that heterotepalins exhibit an high percentage identity with other type 1 RIPs isolated from Phytolaccaceae. Some heterotepalins cross-react with antisera raised against RIPs isolated from Phytolacca dioica leaves. The complete amino acid sequence of heterotepalin 4 matches that of Phytolacca heterotepala anti-viral protein PAP (RIP1), deduced from the cDNA sequence of PhRIP1 gene (AC: AY327475), with one exception concerning residue 245 which, in the native protein, is Ile instead of Met. This substitution, found by mass spectrometry mapping, has been directly confirmed by Edman degradation sequencing of the C-terminal tryptic peptide 242-262. The results show the high potential of mass spectrometry and Edman degradation to verify and to uncover possible amino acid substitutions between native proteins and their cDNA deduced sequences.     

Mood disorder in a patient with Smith-Magenis syndrome: a case report

Smith-Magenis syndrome (SMS) is a microdeletion syndrome characterized by physical and neurobehavioural features. This report describes the case of a 27 year old female affected by SMS associated with a diagnosis, according to DSMIV criteria, of Mood Disorder N.O.S. and Intermittent Explosive Disorder. To our knowledge, the association of SMS with mood shifts has never been reported. Considering the genetic alterations that characterizes the SMS, further investigations on the region of the chromosome 17p11.2 could help produce more information on the role of melatonin in the genesis of mood disorder.     

IL-21 counteracts the regulatory T cell-mediated suppression of human CD4+ T lymphocytes

High expression of IL-21 and/or IL-21R has been described in T cell-mediated inflammatory diseases characterized by defects of counterregulatory mechanisms. CD4(+)CD25(+) regulatory T cells (Treg) are a T cell subset involved in the control of the immune responses. A diminished ability of these cells to inhibit T cell activation has been documented in immune-inflammatory diseases, raising the possibility that inflammatory stimuli can block the regulatory properties of Treg. We therefore examined whether IL-21 controls CD4(+)CD25(+) T cell function. We demonstrate in this study that IL-21 markedly enhances the proliferation of human CD4(+)CD25(-) T cells and counteracts the suppressive activities of CD4(+)CD25(+) T cells on CD4(+)CD25(-) T cells without affecting the percentage of Foxp3(+) cells or survival of Treg. Additionally, CD4(+)CD25(+) T cells induced in the presence of IL-21 maintain the ability to suppress alloresponses. Notably, IL-21 enhances the growth of CD8(+)CD25(-) T cells but does not revert the CD4(+)CD25(+) T cell-mediated suppression of this cell type, indicating that IL-21 makes CD4(+) T cells resistant to suppression rather than inhibiting CD4(+)CD25(+) T cell activity. Finally, we show that IL-2, IL-7, and IL-15, but not IL-21, reverse the anergic phenotype of CD4(+)CD25(+) T cells. Data indicate that IL-21 renders human CD4(+)CD25(-) T cells resistant to Treg-mediated suppression and suggest a novel mechanism by which IL-21 could augment T cell-activated responses in human immune-inflammatory diseases.