Ca2+-Dependent Protein Kinase11 and 24 Modulate the Activity of the Inward Rectifying K+ Channels in Arabidopsis Pollen Tubes

Potassium (K⁺) influx into pollen tubes via K⁺ transporters is essential for pollen tube growth; however, the mechanism by which K⁺ transporters are regulated in pollen tubes remains unknown. Here, we report that Arabidopsis thaliana Ca²⁺-dependent protein kinase11 (CPK11) and CPK24 are involved in Ca²⁺-dependent regulation of the inward K⁺ (K+_in) channels in pollen tubes. Using patch-clamp analysis, we demonstrated that K+_in currents of pollen tube protoplasts were inhibited by elevated [Ca²⁺]_cyt. However, disruption of CPK11 or CPK24 completely impaired the Ca²⁺-dependent inhibition of K+_in currents and enhanced pollen tube growth. Moreover, the cpk11 cpk24 double mutant exhibited similar phenotypes as the corresponding single mutants, suggesting that these two CDPKs function in the same signaling pathway. Bimolecular fluorescence complementation and coimmunoprecipitation experiments showed that CPK11 could interact with CPK24 in vivo. Furthermore, CPK11 phosphorylated the N terminus of CPK24 in vitro, suggesting that these two CDPKs work together as part of a kinase cascade. Electrophysiological assays demonstrated that the Shaker pollen K+_in channel is the main contributor to pollen tube K+_in currents and acts as the downstream target of the CPK11-CPK24 pathway. We conclude that CPK11 and CPK24 together mediate the Ca²⁺-dependent inhibition of K+_in channels and participate in the regulation of pollen tube growth in Arabidopsis.     

DNA Transfection of Bone Marrow Stromal Cells Using Microbubble-Mediated Ultrasound and Polyethylenimine: An In Vitro Study

Non-viral vector transfection efficiency is an issue affecting the clinical application of stem cell gene therapy. This study makes use of the synergistic effect of combining ultrasound (US) with microbubbles (MB) and polyethylenimine (PEI) to increase DNA transfection efficiency, which will enhance the efficiency of gene transfer to bone marrow stromal cells (BMSCs). The optimal parameters for primary-cultured rat-BMSC DNA transfection were examined. The study was arranged based on uniform design. Using a construct containing hepatocyte growth factor (HGF) tagged with enhanced green fluorescent protein (pEGFP-HGF) as example, the mixture of BMSCs, MB, and PEI:DNA complex were exposed to US with frequency of 1 MHz and 10 % duty cycle pulses. Other factors such as acoustic intensity (Q), MB dosage, and total treatment time (T) were also tested. The results were analyzed by regression analysis. Using the best match of parameters, Q = 0.6 W/cm², MB = 10⁶/ml, T = 30 s, different groups were compared. The cooperativity of MB-mediated US and PEI enhanced the gene transfection efficiency by nearly 38-times compared to the DNA without US group. Furthermore, the expression of HGF protein was confirmed by Western blot. The eGFP could be not only seen mainly at the cytoplasm, but also seen in the nucleus in a small proportion of the cells (<10 %) for up to 7 observed days. The transfected BMSCs maintained their capability of multi-directional differentiation and reproductive activity. Our results provide useful information in establishing a novel non-viral transfection method, which may be applied to clinical application in stem cell gene therapy.     

The influence of light conditions and interspecific competition on the root foraging traits and seedling growth of two tree species

Abstract

Enriched nutrient patches within natural soil represent an important source of nutrients for tree growth. In the present study, pot experiments in a heterogeneous nutrient environment were conducted to investigate the influence of light conditions and interspecific competition on the root foraging traits and seedling growth of Pinus massoniana and Schima superba. The root foraging scale and the whole-seedling biomass of both species were decreased by shading. The result of this treatment was a lower sensitivity to nutrient heterogeneity in plants that underwent the shading treatment than in plants that were exposed to full-light conditions. The above-ground biomass and whole-seedling biomass of S. superba were not affected by competition with P. massoniana. In contrast, the above-ground biomass and whole-seedling biomass of P. massoniana were negatively affected by competition with S. superba. The more rapid rate of root extension and the more efficient resource uptake of S.superba appear to explain this effect. The species-specific patterns of the influence of environmental factors on foraging ability and seedling growth should be given thorough consideration and should be applied to afforestation and to the management of tree plantations.     

Derivation and characterization of cell cultures from the skin of the Indo-Pacific humpback dolphin Sousa chinensis

The marine mammalian Indo-Pacific humpback dolphin, once widely lived in waters of the Indian to western Pacific oceans, has become an endangered species. The individual number of this dolphin has significantly declined in recent decades, which raises the concern of extinction. Direct concentration on laboratorial conservation of the genetic and cell resources should be paid to this marine species. Here, we report the successful derivation of cell lines form the skin of Indo-Pacific humpback dolphin. The cell cultures displayed the characteristics of fibroblast in morphology and grew rapidly at early passages, but showed obvious growth arrest at higher passages. The karyotype of the cells consisted of 42 autosomes and sex chromosomes X and Y. The immortalized cell lines obtained by forced expression of the SV40 large T-antigen were capable of proliferation at high rate in long-term culture. Immortalization and long-term culture did not cause cytogenetically observable abnormality in the karyotype. The cell type of the primary cultures and immortalized cell lines were further characterized as fibroblasts by the specific expression of vimentin. Gene transfer experiments showed that exogenetic genes could be efficiently delivered into the cells by both plasmid transfection and lentivirus infection. The cells derived from the skin of the Indo-Pacific humpback dolphin may serve as a useful in vitro system for studies on the effects of environmental pollutants and pathogens in habitats on the dolphin animals. More importantly, because of their high proliferation rate and susceptibility to lentivirus, these cells are potential ideal materials for generation of induced pluripotent stem cells.     

Hydrogen sulfide alleviates uranium-induced kidney cell apoptosis mediated by ER stress via 20S proteasome involving in Akt/GSK-3β/Fyn-Nrf2 signaling

Abstract

Hydrogen sulfide (H₂S) shows antioxidative, anti-inflammatory, antiapoptotic, and cytoprotective effects in kidneys. Recently, H₂S has been reported to alleviate uranium-induced rat nephrotoxicity through oxidative stress and inflammatory response via Nrf2-NF-κB pathways. Here, the protective effect and molecular mechanism of H₂S on uranium-induced apoptosis were examined in normal rat kidney proximal cells (NRK-52^E) in vitro. The results indicate that NaHS (an H₂S donor) administration in uranium-intoxicated kidney cells ameliorated uranium-induced reactive oxygen species generation, caspase-3-dependent apoptosis, and endoplasmic reticulum (ER) stress identified through several key markers including GRP78, C/EBP homologous protein (CHOP), and caspase-12. NaHS treatment in uranium-intoxicated kidney cells abolished the effects of uranium on Akt phosphorylation, GSK-3β activation, increased Fyn nuclear expression, and concomitantly decreased Nrf2 nuclear expression. NaHS administration in uranium-treated kidney cells resorted uranium-decreased the expression of two key subunit PSMA6 and PSMB7 in 20S proteasome. But, DRB (an Nrf2 inhibitor) administration abrogated the effects of NaHS on PSMA6 and PSMB7 expression in uranium-contaminated kidney cells. Bortezomib (a proteasome inhibitor) treatment in NaHS pulsing uranium cotreated kidney cells reversed the effects of NaHS on not only PSMA6 and PSMB7 but also GRP78 and CHOP. Taken together, all data suggest that H₂S can attenuate uranium-induced kidney cell apoptosis mediated by ER stress via 20S proteasome involving in Akt/GSK-3β/Fyn-Nrf2 signaling axis.     

Biosynthesis,characterization and antimicrobial activity of silver nanoparticles by a halotolerant Bacillus endophyticus SCU-L

Abstract

We have conducted a thorough study on extracellular biosynthesis of silver nanoparticles (AgNPs) by a halotolerant bacterium Bacillus endophyticus SCU-L, which was identified by 16S rRNA gene sequencing analysis. This strain was selected during an ongoing research programme aimed at finding a novel biological method for green nanosynthetic routes using the extremophiles in unexplored hypersaline habitats. The biosynthesized AgNPs were characterized and analyzed with UV–vis spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, atomic force microscopy and X-ray diffraction. Further, the AgNPs were found to be spherical in shape with an average particle size of about 5.1?nm, and it was stable in aqueous solution for three months period of storage at room temperature under dark condition. Also, the synthesized AgNPs significantly presented antimicrobial activity against Candida albicans, Escherichia coli, Salmonella typhi and Staphylococcus aureus. The above results suggested that the present work may provide a valuable reference and theoretical basis for further exploration on microbial biosynthesis of AgNPs by halotolerant bacteria.     

ATPase‐driven oligomerization of RIG‐I on RNA allows optimal activation of type‐I interferon

The cytosolic pathogen sensor RIG‐I is activated by RNAs with exposed 5′‐triphosphate (5′‐ppp) and terminal double‐stranded structures, such as those that are generated during viral infection. RIG‐I has been shown to translocate on dsRNA in an ATP‐dependent manner. However, the precise role of the ATPase activity in RIG‐I activation remains unclear. Using in vitro‐transcribed Sendai virus defective interfering RNA as a model ligand, we show that RIG‐I oligomerizes on 5′‐ppp dsRNA in an ATP hydrolysis‐dependent and dsRNA length‐dependent manner, which correlates with the strength of type‐I interferon (IFN‐I) activation. These results establish a clear role for the ligand‐induced ATPase activity of RIG‐I in the stimulation of the IFN response.