A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.     

Structurally different flavonol glycosides and hydroxycinnamic acid derivatives respond differently to moderate UV-B radiation exposure

The aim of this study was to investigate the modifying influence of moderate ultraviolet-B (UV-B) radiation exposure on structurally different flavonol glycosides and hydroxycinnamic acid derivatives during pre-harvest using kale, a leafy Brassica species with a wide spectrum of different non-acylated and acylated flavonol glycosides. Juvenile kale plants were treated with short-term (1 day), moderate UV-B radiation [0.22-0.88 kJ m?2 day?1 biologically effective UV-B (UV-B(BE))]. Twenty compounds were quantified, revealing a structure-specific response of flavonol glycosides and hydroxycinnamic acid derivatives to UV-B radiation. A dose- and structure-dependent response of the investigated phenolic compounds to additional UV-B radiation was found. The investigated quercetin glycosides decreased under UV-B; for kaempferol glycosides, however, the amount of sugar moieties and the flavonol glycoside hydoxycinnamic acid residue influenced the response to UV-B. Monoacylated kaempferol tetraglucosides decreased in the investigated UV-B range, whereas the monoacylated kaempferol diglucosides increased strongly with doses of 0.88 kJ m?2 day?1 UV-B(BE) . The UV-B-induced increase in monoacylated kaempferol triglucosides was dependent on the acylation pattern. Furthermore, the hydroxycinnamic acid glycosides disinapoyl-gentiobiose and sinapoyl-feruloyl-gentiobiose were enhanced in a dose-dependent manner under UV-B. While UV-B radiation treatments often focus on flavonol aglycones or total flavonols, our investigations were extended to structurally different non-acylated and acylated glycosides of quercetin and kaempferol.     

Changes in ect2 localization couple actomyosin-dependent cell shape changes to mitotic progression

As they enter mitosis, animal cells undergo profound actin-dependent changes in shape to become round. Here we identify the Cdk1 substrate, Ect2, as a central regulator of mitotic rounding, thus uncovering a link between the cell-cycle machinery that drives mitotic entry and its accompanying actin remodeling. Ect2 is a RhoGEF that plays a well-established role in formation of the actomyosin contractile ring at mitotic exit, through the local activation of RhoA. We find that Ect2 first becomes active in prophase, when it is exported from the nucleus into the cytoplasm, activating RhoA to induce the formation of a mechanically stiff and rounded metaphase cortex. Then, at anaphase, binding to RacGAP1 at the spindle midzone repositions Ect2 to induce local actomyosin ring formation. Ect2 localization therefore defines the stage-specific changes in actin cortex organization critical for accurate cell division.     

Cell shape: taking the heat

Preservation of cell architecture under physically stressful conditions is a basic requirement for many biological processes and is critical for mechanosensory systems built to translate subtle changes in cell shape into changes in organism behaviour. A new study reveals how an extracellular protein--Spam--helps mechanosensory organs in the fruit fly to withstand the effects of the water loss that accompanies heat shock.     

Optimal concentrations of cryoprotective agents for semen from stallions that are classified 'good' or 'poor' for freezing

Cryopreserved stallion sperm displays a high degree of male-to-male variability with respect to cell viability after thawing. Animals that have semen with low viability after cryopreservation are classified as 'poor' freezers, and when post-thaw viability is high they are designated as 'good' freezers. Cryoprotective agents that are used for cryopreserving stallion sperm include glycerol, ethylene glycol, methyl formamide, and dimethylformamide, and are typically used in concentrations ranging from 1% to 4%. The aim of this study was to evaluate the osmotic stresses that stallion sperm is exposed to during cryopreservation, and to determine if sperm from 'good' and 'poor' freezers show differences in osmotic tolerance limits and in the suitability of cryoprotective agents. Concentrations of 2-3% of the above mentioned cryoprotectants with freezing extender osmolalities ranging from 580 to 895 mOsm kg(-1) showed the highest motility rates after freeze-thaw, both for 'good' and 'poor' freezers, for all cryoprotectants tested with slightly higher values for glycerol. Freeze-thawed semen from 'poor' freezers was found to have a lower percentage of progressively motile sperm compared to that of 'good' freezers. Assessment of plasma and acrosomal membrane integrity after return to isosmotic conditions revealed that cryopreserved sperm from 'poor' freezers showed lower osmotic tolerance limits as compared to sperm from 'good' freezers. Semen from 'poor' freezers that was frozen using freezing extenders supplemented with more then 2% cryoprotectant showed decreased viability and increased acrosome reaction upon return to isoosmotic conditions, whereas 'good' freezers could withstand cryoprotectant concentrations up to 3% before a decline in viability was observed.