Varied approach of using MSCs for bovine embryo in vitro culture

The objective of the present study was to evaluate the effect of porcine Mesenchymal Stem Cells (MSCs) secreted factors on bovine in vitro embryo development by using MSCs in different culture systems: SOF medium, SOF medium conditioned by MSCs in monolayer and in reverse drop and by embryo culture in co-culture with MSCs. Statistically highly significant differences were noted between the number of blastocysts derived cultures in all tested culture systems. The in vitro culture in SOF turned out to be the most optimal. Statistically highly significant differences were observed in the number of blastocyst obtained between SOF and SOF in co-culture with MSCs (p?<?0.0001), and between SOF and SOF conditioned (monolayer and drop) (p?<?0.00001). The trials to produce blastocysts in SOF conditioned by MSCs in reverse drops and monolayer failed. The blastocysts were obtained and analysed by TUNEL only in two out of four experimental groups: SOF and SOF in co-culture with MSCs. There were no significant differences between any of analysed blastocysts’ groups neither in the total number of nuclei nor in the apoptotic features. Neither medium conditioning by MSCs in monolayer and in reverse drop nor embryo culture in co-culture with MSC turned out to be effective.     

Antimicrobial and antioxidative potential of free and immobilised cellobiose dehydrogenase isolated from wood degrading fungi

Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is a glycoprotein having many biotechnological applications. In the present study, CDHs isolated from Phlebia lindtneri (PlCDH), Phanerochaete chrysosporium (PchCDH), Cerrena unicolor (CuCDH), and Pycnoporus sanguineus (PsCDH) were studied the first time for their ability to generate antioxidant and antimicrobial agents. The aim of the research was to evaluate the antioxidant and antimicrobial activity of systems composed of four CDHs and lactose or cellobiose as a reaction substrate. The free radical scavenging effect of free and immobilised enzymes was evaluated using the DPPH method. The lowest values of EC₅₀ (10.04 ± 0.75 $\text{μg}$/ml) was noted for PlCDH/lactose and for PlCDH/cellobiose (12.06 ± 1.35 $\text{μg}$/ml). The EC₅₀value reached 12.6 ± 1.51 $\text{μg}$/ml in the case of PsCDH/lactose and 15.96 ± 1.35 for PsCDH. The CDH preparations were also effectively immobilised in alginate (the immobilisation efficiency expressed as a protein yield ranged from 61.6 to 100 %). The operational stability expressed as a scavenging effect showed the possibility of using the alginate beads 4 times. Both the free and immobilised CDHs as well as the CDH/substrate were tested against Gram-negative Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Gram-positive Staphylococcus aureus ATCC 25923 bacteria. All samples, except PlCDH, were potentially effective in suppression of bacterial growth. The highest percentage of inhibition (100 %) was obtained for S. aureus bacteria using PsCDH and PchCDH with lactose as a substrate, whereas a slightly lesser effect was observed for E. coli and P. aeruginosa bacterial cells, i.e. 64.1 % and 86.5 % (PsCDH) and 94.1 % and 41.4 % (PchCDH), respectively. Furthermore, the concentrations of the reaction products (aldonic acids and hydrogen peroxide) were quantified and the surface morphology of the alginate beads was analysed using SEM visualisation.     

Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells

We characterized the expression and functional properties of the ADP-sensitive P2Y(1) and P2Y(12) nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y(12) receptor relative to P2Y(1) was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y(1) receptor was low, and the P2Y(12) receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y(12) receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y(12) receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y(1) receptor, indicating the inhibitory role of P2Y(1) in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y(1) to P2Y(12) would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation.     

<Emphasis Type="SmallCaps">l</Emphasis>-Arginine Suppresses Aggregation of Recombinant Growth Hormones in Refolding Process from <Emphasis Type="Italic">E. coli</Emphasis> Inclusion Bodies

l-Arginine was used to suppress the aggregation of recombinant mink and porcine growth hormones in the refolding process from E. coli inclusion bodies by solubilization–dilution protocol at high protein concentration and pH 8.0. The influence of l-arginine concentration on the renaturation yield of both proteins was investigated. l-Arginine effectively suppressed the precipitation of growth hormones during dilution, but did not inhibit soluble oligomers formation. The results of mink and porcine growth hormones purification from 4 g of biomass are presented.     

Application of the salt stress to the protoplast cultures of the carrot (Daucus carota L.) and evaluation of the response of regenerants to soil salinity

This is the first study to generate carrot plants for enhanced salinity tolerance using a single-cell in vitro system. Protoplasts of three carrot accessions were exposed to treatment by seven different concentrations of NaCl (10–400 mM). Salt concentrations higher than 50 mM decreased plating efficiency and those of 200–400 mM of NaCl completely arrested mitotic divisions of cultured cells. The protoplast-derived plants from the control and 50–100 mM NaCl treatment were subjected to an 8-week salt stress in greenhouse conditions induced by salinized soil (EC 3 and 6 mS cm^?1). 50 mM NaCl stress applied in vitro induced polyploidy among regenerated plants. The regenerants obtained from the 50 and 100 mM NaCl-treated protoplast cultures grown in saline soil had a higher survival rate compared to the regenerants from the control cultures. The salt-stressed plants accumulated anthocyanins in petioles and produced denser hairs on leaves and petioles in comparison to the control plants. Salt stress influenced pollen viability and seed setting of obtained regenerants. The results suggest that salt stress applied in vitro in protoplast cultures creates variation which allows alleviating the negative effects of salt stress on the development and reproduction of the carrot.

     

Giant clams discriminate threats along a risk gradient and display varying habituation rates to different stimuli

It is often beneficial for animals to discriminate between different threats and to habituate to repeated exposures of benign stimuli. While much is known about risk perception in vertebrates and some invertebrates, risk perception in marine invertebrates is less extensively studied. One method to study risk perception is to habituate animals to a series of exposures to one stimulus, and then present a novel stimulus to test if it transfers habituation. Transfer of habituation is seen as a continued decrease in response while lack of transfer is seen either by having a similar or greater magnitude response. We asked whether giant clams (Tridacna maxima) discriminate between biologically relevant types of threats along a risk gradient. Giant clams retract their mantle and close their shell upon detecting a threat. While closed, they neither feed nor photosynthesize, and prior work has shown that the cost of being closed increases as the duration of their response increases. We recorded a clam's latency to emerge after simulated threats chosen to represent a risk gradient: exposure to a small shading event, a medium shading event, a large shading event (chosen to simulate fish swimming above them), tapping on their shell and touching their mantle (chosen to simulate different degrees of direct attack). Although these stimuli are initially perceived as threatening, we expected clams to habituate to them because they are ultimately non‐damaging and it would be costly for clams to remain closed for extended periods of time when there is no threat present. Clams had different initial latencies to emerge and different habituation rates to these treatments, and they did not transfer habituation to higher risk stimuli and to some lower risk stimuli. These results suggest that clams discriminated between these stimuli along a risk gradient and the lack of habituation transfer shows that the new stimulus was perceived as a potential threat. This study demonstrates that sessile bivalves can discern between levels of predatory threat. These photosynthetic clams may benefit from being able to categorize predator cues for efficient energy allocation.     

Phosphatidylinositol 4-OH kinase is a downstream target of neuronal calcium sensor-1 in enhancing exocytosis in neuroendocrine cells

Neuronal calcium sensor-1 (NCS-1), the mammalian orthologue of frequenin, belongs to a family of EF-hand-containing Ca(2+) sensors. NCS-1/frequenin has been shown to enhance synaptic transmission in PC12 cells and Drosophila and Xenopus, respectively. However, the precise molecular mechanism for the enhancement of exocytosis is largely unknown. In PC12 cells, NCS-1 potentiated exocytosis evoked by ATP, an agonist to phospholipase C-linked receptors, but had no effect on depolarization-evoked release. NCS-1 also enhanced exocytosis triggered by ionomycin, a Ca(2+) ionophore that bypasses K(+) and Ca(2+) channels. Overexpression of NCS-1 caused a shift in the dose-response curve of inhibition of ATP-evoked secretion using phenylarsine oxide, an inhibitor of phosphatidylinositol 4-OH kinase (PI4K). Plasma membrane phosphatidylinositol 4,5-bisphosphate pools were increased upon NCS-1 transfection as visualized using a phospholipase C-delta pleckstrin homology domain-green fluorescent protein construct. NCS-1-transfected cell extracts displayed increased phosphatidylinositol-4-phosphate biosynthesis, indicating an increase in PI4K activity. Mutations in NCS-1 equivalent to those that abolish the interaction of recoverin, another EF-hand-containing Ca(2+) sensor, with its downstream target rhodopsin kinase, lost their ability to enhance exocytosis. Taken together, the present data indicate that NCS-1 modulates the activity of PI4K, leading to increased levels of phosphoinositides and concomitant enhancement of exocytosis.     

Effect of cellularity on the sensitivity of detecting squamous lesions in liquid-based cervical cytology

OBJECTIVE: To evaluate the effect of cellularity on the sensitivity of both screening and diagnosis in a liquid-based cervical sample. STUDY DESIGN: SurePath samples (TriPath Imaging Inc., Burlington, North Carolina, U.S.A.) with known diagnoses were selected, including 18 negative, 16 low grade squamous intraepithelial lesion (LSIL) and 12 high grade squamous intraepithelial lesion (HSIL) cases. Through a serial dilution technique, samples of varying cellularity were prepared. The 275 slides were assigned random numbers and were routinely screened by 1 of 2 senior cytotechnologists, blinded to the reference diagnosis. Specimens with a screening diagnosis of atypical squamous cells of undetermined significance (ASCUS) or higher were reviewed by two pathologists, resulting in a final consensus diagnosis. Using a grid counting system, cellularity was determined for each slide. RESULTS: There was a clear demarcation in sensitivity between specimens with a cellularity of < 5,000 or > or = 5,000 squamous cells. This applied to both the sensitivity for screening and to the final consensus diagnosis. For cases with a reference diagnosis of LSIL+, at a cytotechnologist screening level of ASCUS or greater, sensitivity increased from 72.8% (< 5,000 cells) to 98.1% (> or = 5,000 cells) and for a reference diagnosis of HSIL from 85.7% to 100%, respectively. Similarly, for the consensus diagnosis, sensitivity rose from 78.5% (< 5,000 cells) to 96.6% (> or = 5,000 cells) for LSIL+ and from 82.9% to 100%, respectively, for HSIL. These differences were statistically significant (P < .001). CONCLUSION: A minimum cellularity of 5,000 squamous cells is recommended for SurePath liquid-based cervical preparations.     

Low temperature-induced modifications in cell ultrastructure and localization of phenolics in winter oilseed rape (Brassica napus L. var. oleifera L.) leaves

Acclimation of winter oilseed plants in the cold (i.e. at temperatures >0 degrees C) followed by short exposure to sub-lethal freezing temperatures resulted in pronounced ultrastructural changes of leaf epidermal and mesophyll cells. The following major changes were observed upon acclimation at 2 degrees C: increased thickness of cell walls; numerous invaginations of plasma membranes; the appearance of many large vesicles localized in the cytoplasm in close proximity to the central vacuole; the occurrence of abundant populations of microvesicles associated with the endoplasmic reticulum (ER) cisternae or located in the vicinity of dictyosomes; and the occurrence of paramural bodies and myelin-like structures. In addition, large phenolic deposits were observed in the vicinity of the plasma membrane and membrane-bound organelles such as chloroplasts, large vesicles or cytoplasm/tonoplast interfaces. Transient freezing (-5 degrees C for 18 h) of the cold-acclimated leaves led to reversible disorganization of the cytoplasm and to pronounced structural changes of the cellular organelles. Chloroplasts were swollen, with the stroma occupying one half of their volume and the thylakoid system being displaced to the other half. Large phenolic aggregates disappeared but distinct layers of phenolic deposits were associated with mitochondrial membranes and with chloroplast envelopes. In frost-thawed cells recovered at 2 degrees C for 24 h, dictyosomes and dictyosome- or ER-derived small vesicles reappeared in the ribosome-rich cytoplasm. Aberrations in the structure of chloroplasts and mitochondria were less pronounced. Few phenolic deposits were seen as small grains associated with chloroplast envelopes and vesicle membranes. These observations demonstrate that plants undergo different changes in cell ultrastructure depending on whether they are subjected to chilling or freezing temperatures. Results are discussed in relation to membrane recycling and the possible role of phenolics during the first and second stages of plant acclimation at low temperature.