Inhibitory effect of aspirin on cholera toxin-induced phospholipase and cyclo-oxygenase activity

Cholera toxin (CT) stimulated phospholipase activity and caused [3H]arachidonic acid (3H-AA) release in a murine macrophage/monocyte cell line. Pretreatment of cells with dexamethasone, a phospholipase A2 (PLA2) inhibitor, did not affect CT-induced 3H-AA release. In contrast, aspirin, which is an inhibitor of phospholipase C (PLC), blocked CT-induced 3H-AA release and subsequent prostaglandin (PC) synthesis. The inhibitory effect of aspirin was dose dependent, with 4 mM reducing the CT response by approximately 50%. Similarly, inhibition was time dependent, occurring when the drug was added to the culture medium as late as 30 min after CT. Brief exposure (30 min) of the cells to aspirin did not alter their subsequent response to CT, but 3H-AA release from cells exposed to aspirin for 2.5 h was irreversibly inhibited. The data suggested that CT stimulation of AA metabolism may involve increased PLC activity.     

Methylation of KvDMR1 involved in regulating the imprinting of CDKN1C gene in cattle

The CDKN1C gene encodes a cyclin‐dependent kinase inhibitor and is one of the key genes involved in the development of Beckwith–Wiedemann syndrome and cancer. In this study, using a direct sequencing approach based on a single nucleotide polymorphism (SNP) at genomic DNA and cDNA levels, we show that CDKN1C exhibits monoallelic expression in all seven studied organs (heart, liver, spleen, lung, kidney, muscle and subcutaneous fat) in cattle. To investigate how methylation regulates imprinting of CDKN1C in cattle, allele‐specific methylation patterns in two putative differential methylation regions (DMRs), the CDKN1C DMR and KvDMR1, were analyzed in three tissues (liver, spleen and lung) using bisulfite sequencing PCR. Our results show that in the CDKN1C DMR both parental alleles were unmethylated in all three analyzed tissues. In contrast, KvDMR1 was differentially methylated between the two parental alleles in the same tissues. Statistical analysis showed that there is a significant difference in the methylation level between the two parental alleles (P < 0.01), confirming that this region is the DMR of KvDMR1 and that it may be correlated with CDKN1C imprinting.     

Rice G protein γ subunit qPE9-1 modulates root elongation for phosphorus uptake by involving 14-3-3 protein OsGF14b and plasma membrane H+-ATPase

Heterotrimeric G protein is involved in plant growth and development, while the role of rice (Oryza sativa) G protein γ subunit qPE9-1 in response to low-phosphorus (LP) conditions remains unclear. The gene expression of qPE9-1 was significantly induced in rice roots under LP conditions. Rice varieties carrying the qPE9-1 allele showed a stronger primary root response to LP than the varieties carrying the qpe9-1 allele (mutant of the qPE9-1 allele). Transgenic rice plants with the qPE9-1 allele had longer primary roots and higher P concentrations than those with the qpe9-1 allele under LP conditions. The plasma membrane (PM) H⁺-ATPase was important for the qPE9-1-mediated response to LP. Furthermore, OsGF14b, a 14-3-3 protein that acts as a key component in activating PM H⁺-ATPase for root elongation, is also involved in the qPE9-1 mediation. Moreover, the overexpression of OsGF14b in WYJ8 (carrying the qpe9-1 allele) partially increased primary root length under LP conditions. Experiments using R18 peptide (a 14-3-3 protein inhibitor) showed that qPE9-1 is important for primary root elongation and H⁺ efflux under LP conditions by involving the 14-3-3 protein. In addition, rhizosheath weight, total P content, and the rhizosheath soil Olsen-P concentration of qPE9-1 lines were higher than those of qpe9-1 lines under soil drying and LP conditions. These results suggest that the G protein γ subunit qPE9-1 in rice plants modulates root elongation for phosphorus uptake by involving the 14-3-3 protein OsGF14b and PM H⁺-ATPase, which is required for rice P use.     

Mediator structure and conformation change

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Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.     

Enhanced viability of Lactobacillus reuteri for probiotics production in mixed solid-state fermentation in the presence of Bacillus subtilis

In order to develop a multi-microbe probiotic preparation of Lactobacillus reuteri G8-5 and Bacillus subtilis MA139 in solid-state fermentation, a series of parameters were optimized sequentially in shake flask culture. The effect of supplementation of B. subtilis MA139 as starters on the viability of L. reuteri G8-5 was also explored. The results showed that the optimized process was as follows: water content, 50 %; initial pH of diluted molasses, 6.5; inocula volume, 2 %; flask dry contents, 30～35 g/250 g without sterilization; and fermentation time, 2 days. The multi-microbial preparations finally provided the maximum concentration of Lactobacillus of about 9.01?±?0.15 log CFU/g and spores of Bacillus of about 10.30?±?0.08 log CFU/g. Compared with pure fermentation of L. reuteri G8-5, significantly high viable cells, low value of pH, and reducing sugar in solid substrates were achieved in mixed fermentation in the presence of B. subtilis MA139 (P?<?0.05). Meanwhile, the mixed fermentation showed the significantly higher antimicrobial activity against E. coli K88 (P?<?0.05). Based on the overall results, the optimized process enhanced the production of multi-microbe probiotics in solid-state fermentation with low cost. Moreover, the viability of L. reuteri G8-5 could be significantly enhanced in the presence of B. subtilis MA139 in solid-state fermentation, which favored the production of probiotics for animal use.     

A comparative analysis of photosynthetic characteristics of hulless barley at two altitudes on the Tibetan Plateau

To determine the photosynthetic characteristics of C₃ plants and their sensitivity to CO₂ at different altitudes on the Tibetan Plateau, hulless barley (Hordeum vulgare L. ssp. vulgare) was grown at altitudes of 4,333 m and 3,688 m. Using gas-exchange measurements, photosynthetic parameters were simulated, including the maximum net photosynthesis (P _max) and the apparent quantum efficiency (α). Plants growing at higher altitude had higher net photosynthetic rates (P _N), photosynthesis parameters (P _max and α) and sensitivities to CO₂ enhancement than plants growing at lower altitude on the Tibetan Plateau. The enhancements of P _N, P _max, and α for plants growing at higher altitude, corresponding with 10 μmol(CO₂) mol⁻¹ increments, were approximately 0.20∼0.45%, 0.05∼0.20% and 0.12∼0.36% greater, respectively, than for plants growing at lower altitude, respectively, where CO₂ levels rose from 10 to 170 μmol(CO₂) mol⁻¹. Therefore, on the Tibetan Plateau, the changes in the photosynthetic capacities and the photosynthetic sensitivities to CO₂ observed in the C₃ plants grown above 3,688 m are likely to increase with altitude despite the decreasing CO₂ partial pressure.