Optimization of conditions for germination of cold-stored <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> pollen

One of the rare weak points of the model plant Arabidopsis is the technical problem associated with the germination of its male gametophyte and the generation of the pollen tube in vitro. Arabidopsis pollen being tricellular has a notoriously low in vitro germination compared to species with bicellular pollen. This drawback strongly affects the reproducibility of experiments based on this cellular system. Together with the fact that pollen collection from this species is tedious, these are obstacles for the standard use of Arabidopsis pollen for experiments that require high numbers of pollen tubes and for which the percentage of germination needs to be highly reproducible. The possibility of freeze-storing pollen after bulk collection is a potential way to solve these problems, but necessitates methods that ensure continued viability and reproducible capacity to germinate. Our objective was the optimization of germination conditions for Arabidopsis pollen that had been freeze-stored. We optimized the concentrations of various media components conventionally used for in vitro pollen germination. We found that in general 4 mM calcium, 1.62 mM boric acid, 1 mM potassium, 1 mM magnesium, 18% sucrose at pH 7 and a temperature of 22.5°C are required for optimal pollen germination. However, different experimental setups may deviate in their requirements from this general protocol. We suggest how to optimally use these optimized methods for different practical experiments ranging from morphological observations of pollen tubes in optical and electron microscopy to their bulk use for molecular and biochemical analyses or for experimental setups for which a specific medium stiffness is critical. F. Bou Daher and Y. Chebli contributed equally to this study.     

Isolation and partial characterization of three pregnancy-associated glycoproteins from the ewe placenta

Pregnancy-associated glycoproteins (PAGs) are synthesized in the outer epithelial layer of the placenta in artiodactyls. In this work, three novel ovine PAGs were isolated from late-pregnancy fetal cotyledons and characterized biochemically. The isolation procedure included acid and ammonium sulfate precipitations and anion and cation exchange chromatographies. The isolated PAGs have different NH(2)-terminal amino acid sequences (RGSXLTILPLRNMRDIVY, ISRVSXLTIHPLRNIMDML, and RGSNLTIHPLRNIRD) and apparent molecular masses (55, 57, and 59 kDa). Each shows several isoforms with different pI values. The three proteins share high sequence identity with each other and with other ovine, bovine, and caprine PAGs. They have not been described previously. The ovPAG-59 sequence differs from the previously identified ovPAG-4 sequence (determined by DNA cloning and sequencing) at only one position among the 15 N-terminal residues. The newly characterized ovPAGs and the procedure used to isolate them will be helpful in producing new antisera for investigating PAG secretion in pregnant ewes.     

Pregnancy-associated glycoprotein concentrations during pregnancy and the postpartum period in Azawak Zebu cattle

Specific RIA systems were developed and used to measure pregnancy-associated glycoprotein (PAG) concentrations during gestation and the postpartum period in Azawak Zebu cows. Twelve females were palpated per rectum and diagnosed as pregnant. Blood samples were taken at 5-10-day intervals from approximately Week 8 of gestation until Week 10 postpartum (pp). One Zebu cow (Z15) initially diagnosed as pregnant showed PAG concentrations lower than the assay sensitivity (<0.20 ng/ml) and did not calve. Another cow (ZSand) showed abnormally high PAG concentrations during gestation and was excluded from the general PAG profile. The 10 other Zebu cows exhibited a very similar PAG profile. In these animals, concentrations increased progressively from Week 8 to 35 of gestation (from 6.0+/-4.2 to 196.0+/-34.8 ng/ml), remaining relatively constant until Week 39 (210.8+/-74.8 ng/ml), when they increased sharply to reach their highest level (1095.6+/-607.2 ng/ml) at around parturition. After delivery, PAG concentrations declined significantly (P<0.05) until Week 2 postpartum (348.4+/-85.6 ng/ml) and slowly until Week 10 postpartum. Our results revealed that the PAG pattern in Zebu cattle was similar to those of taurine breeds during the first two trimesters of pregnancy, but differed in the peripartum period.     

The RasGAP-associated endoribonuclease G3BP assembles stress granules

Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents, and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149, whose dephosphorylation is induced by arsenite treatment. Critically, a phosphomimetic mutant (S149E) fails to oligomerize and to assemble SGs, whereas a nonphosphorylatable G3BP mutant (S149A) does both. These results suggest that G3BP is an effector of SG assembly, and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.     

Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

Background

Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K⁺ channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before.

Results

Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair.

Conclusion

Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.     

Comparison between the recovery rate of three concentration protocols of water samples intended for analysis by Molecular Biology: Membrane filtration,filtration on gauze pad and centrifugation

The presence of microorganisms (bacteria, protozoa, viruses, etc.) in water is a crucial indicator of its quality and safety. The detection of these microorganisms by conventional and classical techniques is widely used in water quality control laboratories; nevertheless these methods have limitations in terms of rapidity and precision of results. The use of Molecular Biology has been a great evolution in the techniques of water analysis. However, the choice of the concentration protocol allowing for the best rate of microorganism recovery in a suspension remains a real challenge. The objective of this experimental study is to compare the recovery rate of three different protocols of water concentration (membrane filtration, filtration on gauze pad and centrifugation) for samples intended for analysis by polymerase chain reaction PCR. Which can then serve as a reference protocol for water quality control laboratories. The experimental results have shown that the membrane filtration protocol yields the best recovery rate and concentration of microorganisms followed by filtration on gauze pad, while the centrifugation protocol (8000g, 10 min, 22 °C) gives the lowest rate of recovery out of the three protocols. The experimental results obtained through this study allows us to contribute to the optimization and standardization of water samples concentration techniques intended for analysis by Molecular Biology.     

Potentiation of developmental abilities of diapausing eggs of Bombyx mori by 20-hydroxyecdysone

Diapausing Bombyx mori eggs were treated with 20-hydroxyecdysone as follows: a 30 minute immersion in bleaching water was followed by a 24-hour soaking in a 10^?4 M solution of the hormone. Of the treated embryos 85% developed to some extent and 25% of them terminated their embryonic development. The efficiency of the treatment rapidly decreased when eggs were allowed to hibernate at 7°C in order to break diapause.     

Alterations of hepatocyte function with free radical generators and reparation or prevention with coffee polyphenols

Liver diseases are linked in the majority of cases to oxidative stress that antioxidants could neutralize with reducing liver injury. Chlorogenic acid, a coffee polyphenol, possesses antioxidant prosperities. The aim of this study was to evaluate in vitro preventive and corrective effects of cholorogenic acid in hepatocyte toxicity induced by free radicals. Hepatocytes were isolated from adult male Wistar rats. To determine corrective effects and reparation, cells were first exposed to two free radical generators (hydrogen peroxide/iron sulfate for hydroxyl radical formation, and phenazine methosulfate/nicotinamide adenine dinucleotide for superoxide anion formation) for 12H and thereafter treated by chlorogenic acid (1 and 10?μM final concentration) for another 12H. To show preventive effects, cells were pretreated by chlorogenic acid and thereafter exposed to free radical generators. Hepatocyte proliferation, glucose uptake, ATP contents, membrane fluidity and integrity, and intracellular redox status were investigated after 24H culture. The results showed that chlorogenic acid reversed the decrease in cell proliferation, glucose uptake and ATP levels, the increased LDH release and the reduced membrane fluidity and restored the oxidant/antioxidant status under oxidative stress. When pre-treated with chlorogenic acid, hepatocytes became very resistant to oxidative conditions and cellular homeostasis was maintained. In conclusion, chlorogenic acid displayed not only corrective but also preventive effects in hepatocytes exposed to oxidative stress and could be beneficial in patients with or at risk of liver diseases.