Kinase SnRK1.1 regulates nitrate channel SLAH3 engaged in nitrate-dependent alleviation of ammonium toxicity

Nitrate (NO3−) and ammonium (NH4+) are major inorganic nitrogen (N) supplies for plants, but NH4+ as the sole or dominant N source causes growth inhibition in many plants, known as ammonium toxicity. Small amounts of NO3− can significantly mitigate ammonium toxicity, and the anion channel SLAC1 homolog 3 (SLAH3) is involved in this process, but the mechanistic detail of how SLAH3 regulates nitrate-dependent alleviation of ammonium toxicity is still largely unknown. In this study, we identified SnRK1.1, a central regulator involved in energy homeostasis, and various stress responses, as a SLAH3 interactor in Arabidopsis (Arabidopsis thaliana). Our results suggest that SNF1-related protein kinase 1 (SnRK1.1) functions as a negative regulator of SLAH3. Kinase assays indicate SnRK1.1 strongly phosphorylates the C-terminal of SLAH3 at the site S601. Under high-NH4+/low-pH condition, phospho-mimetic and phospho-dead mutations in SLAH3 S601 result in barely rescued phenotypes and fully complemented phenotypes in slah3. Furthermore, SnRK1.1 migrates from cytoplasm to nucleus under high-NH4+/low-pH conditions. The translocation of SnRK1.1 from cytosol to nucleus under high-ammonium stress releases the inhibition on SLAH3, which allows SLAH3-mediated NO3− efflux leading to alleviation of high-NH4+/low-pH stress. Our study reveals that the C-terminal phosphorylation also plays important role in SLAH3 regulation and provides additional insights into nitrate-dependent alleviation of ammonium toxicity in plants.

Nitrate-dependent alleviation of ammonium toxicity involves negative regulation of a nitrate channel by a kinase.     

Effect of various glucose/glutamine ratios on hybridoma growth,viability and monoclonal antibody formation

Summary Cultivation of a model hybridoma cell line in Dulbecco's modification of Eagle's minimal essential medium (DMEM) at various glucose and glutamine levels showed that maxmium cell growth was obtained in a broad range of glucose/glutamine ratios of 0.6 to 6.0. A new parameter called the viability index is proposed to quantify cell viability. A strong correlation between viability index and monoclonal antibody (MAb) concentration was found at all glucose/glutamine ratios investigated.     

Yeast calmodulin: structural and functional differences compared with vertebrate calmodulin

Calmodulin of the baker's yeast (Saccharomyces cerevisiae) showed a similar affinity for Ca2+ to that of vertebrate calmodulin. The maximum binding number of Ca2+ to yeast calmodulin was, however, 3 mol/mol, which is lower than that of vertebrate calmodulin (4 mol/mol). The same maximum activity of porcine brain phosphodiesterase was attained when 100 times higher concentration of yeast calmodulin than that of vertebrate calmodulin was added. On the other hand, the maximum activation of chicken gizzard myosin light chain kinase was attained with 1,000 times higher concentration of yeast calmodulin than that of vertebrate calmodulin, and the maximum activity with yeast calmodulin was less than 1/5 of that with vertebrate calmodulin. Several amino acid substitutions observed in the yeast calmodulin, particularly at the alpha-helical rod connecting the two globular domains, may affect the interaction mode of various target enzymes with this calmodulin.     

酿酒酵母转化木糖生产化学品的研究进展

木糖的有效利用是木质纤维素生产生物燃料或化学品经济性转化的基础.30年来,通过理性代谢改造和适应性进化等工程策略,显著提高了传统乙醇发酵微生物——酿酒酵母Saccharomyces cerevisiae的木糖代谢能力.因此,近年来在酿酒酵母中利用木糖生产化学品的研究逐步展开.研究发现,酿酒酵母分别以木糖和葡萄糖为碳源时...     

Dielectric spectroscopy of L-alpha-lysolecithin-packaged gramicidin A

The complex permittivities of L-alpha-lysolecithin in the absence and presence of the gramicidin A ion channel were measured over the temperature range 0-60 degrees C and over the frequency range 1-1000 MHz. One dielectric relaxation/loss has been observed. It is located at 103.3 MHz (1.54 ns) for a micellar 0.4 M L-alpha-lysolecithin solution at 20 degrees C, whereas it is shifted to 71.7 MHz (2.22 ns) for a lamellar L-alpha-lysolecithin-gramicidin A aqueous solution (0.4 M L-alpha-lysolecithin, 0.0308 M gramicidin A) at 20 degrees C. The dielectric relaxation decreases and the relaxation time increases when gramicidin A is incorporated into L-alpha-lysolecithin. These dielectric changes are related, in part, to the micellar-to-lamellar lipid phase transition induced by the incorporation of gramicidin A into lysolecithin. We suggest that the diffuse rotational motion of the polar head group of L-alpha-lysolecithin contributes to the dielectric relaxation/loss at around 100 MHz.     

Pantothenate mediates the coordination of whitefly and symbiont fitness

Intracellular symbionts in insects often have reduced genomes. Host acquisition of genes from bacteria is an important adaptation that supports symbionts. However, the function of horizontally transferred genes in insect symbiosis remains largely unclear. The primary symbiont Portiera housed in bacteriocytes lacks pantothenate synthesis genes: panB and panC, which is presumably complemented by a fused gene panB-panC (hereafter panBC) horizontally transferred from bacteria in Bemisia tabaci MEAM1. We found panBC in many laboratory cultures, and species of B. tabaci shares a common evolutionary origin. We demonstrated that complementation with whitefly panBC rescued E. coli pantothenate gene knockout mutants. Portiera elimination decreased the pantothenate level and PanBC abundance in bacteriocytes, and reduced whitefly survival and fecundity. Silencing PanBC decreased the Portiera titer, reduced the pantothenate level, and decreased whitefly survival and fecundity. Supplementation with pantothenate restored the symbiont titer, PanBC level, and fitness of RNAi whiteflies. These data suggest that pantothenate synthesis requires cooperation and coordination of whitefly PanBC expression and Portiera. This host–symbiont co-regulation was mediated by the pantothenate level. Our findings demonstrated that pantothenate production, by the cooperation of a horizontally acquired, fused bacteria gene and Portiera, facilitates the coordination of whitefly and symbiont fitness. Thus, this study extends our understanding on the basis of complex host–symbiont interactions.Subject terms: Applied microbiology, Functional genomics     

Factors governing lactic acid formation in long term cultivation of hybridoma cells

Summary Effect of glucose concentration and pH on lactic acid formation was investigated in batch cultures of a hybridoma cell line. Lactate formation increases with growth rate. High glucose concentration leads to extensive lactate formation only during growth phase and not during stationary phase. Lactate formation also may serve to regulate extracellular pH to pH 6.8, provided conditions are favorable to maintain viability and if sufficient nutrients are present in the medium.     

脐带间充质干细胞治疗熊去氧胆酸应答不佳的原发性胆汁性胆管炎的临床观察

目的探讨脐带间充质干细胞（UC-MSCs）输注治疗熊去氧胆酸（UDCA）应答不佳的原发性胆汁性胆管炎（PBC）患者的安全性和有效性，分析影响UC-MSCs疗效应答的相关因素。 方法选取解放军总医院第五医学中心2010年8月至2017年10月接受UC-MSCs输注治疗UDCA应答不佳的PBC患者29例。患者均以4周为间隔给予3次外周静脉输注细胞1.0×10⁶个/kg。通过实验室指标、生命体征及不良事件发生情况评估UC-MSCs治疗的安全性。通过患者临床症状、肝功指标和Child-Pugh评分评估治疗的有效性。以"巴黎Ⅰ标准"作为疗效标准，评价患者UC-MSCs治疗后的疗效应答情况，比较有效患者及无效患者基线临床症状和肝脏功能差异，分析影响UC-MSCs疗效的相关因素。采用独立样本t检验分析年龄；采用Mann-Whitney U检验比较两组UDCA治疗时间、激素治疗时间、实验室数据等，采用Wilcoxon秩和检验比较组间数据；采用χ²检验比较性别、临床症状和Child-Pugh分级等指标。多因素Cox回归分析对影响UC-MSCs疗效的相关因素。 结果1例患者在治疗后因合并严重感染出现高热，所有患者未出现UC-MSCs相关严重不良事件。UC-MSCs输注后与基线相比，患者的血清碱性磷酸酶（ALP）[281.00 （182.50，428.50）比201.00 （149.50，402.00）]、γ-谷氨酰转移酶（GGT）[156.00 （73.00，390.00）比84.00 （43.50，312.50）]、总胆固醇（TC）[5.10 （3.14，7.69）比3.94 （3.00，6.01）]均下降（P < 0.05）。其中，9例（31﹪）患者治疗后疗效明显，达到"巴黎Ⅰ标准"，与无效组患者基线相比，有效组患者天冬氨酸转氨酶（AST）[93.50 （77.75，100.75）比53.00 （46.00，78.00）]、ALP[342.00 （237.25，516.00）比185.00 （152.50，295.50）]、总胆红素（TBIL）[58.50 （33.45，69.33）比13.10 （11.25，20.25）]均下降（P < 0.05）。多因素Cox回归分析显示，血清TBIL是影响UC-MSCs疗效的重要独立因素[HR为0.817 （95﹪CI：0.715 ~ 0.935），P < 0.05]。 结论UC-MSCs输注对UDCA治疗应答不佳的PBC患者是安全可行的且耐受性良好，部分患者肝功能得到一定的改善。血清TBIL是影响UC-MSCs治疗疗效的独立重要因素，提示在疾病进展早期TBIL较低的阶段进行UC-MSCs治疗可能有效改善和减缓患者疾病进程。     

Molecular characterization of a plant FKBP12 that does not mediate action of FK506 and rapamycin

Immuonosuppressive drugs FK506 and rapamycin block a number of signal transduction pathways in eukaryotic systems. The 12 kDa FK506 binding protein (FKBP12) mediates the action of both FK506 and rapamycin against their functional targets. In this report, we cloned, sequenced and characterized a gene encoding FKBP12 in Vicia faba ( Vf FKBP12). While Vf FKBP12 is highly homologous to animal and yeast FKBP12, it does not mediate the action of FK506 and rapamycin. There are unique features in plant FKBP12 sequences that cause the variation in their function. One lies in the domain that is critical for interaction with calcineurin (CaN), the mammalian and yeast target of FKBP12-FK506 complex. Protein–protein interaction assays revealed a low-affinity and unstable Vf FKBP12-FK506-CaN ternary complex. In the genetic assay, Vf FKBP12 did not restore the sensitivity of yeast FKBP12 mutant to rapamycin or FK506, supporting that plant FKBP12-ligand complexes are unable to block the function of the drug target. Also unique to plant FKBP12 proteins, a pair of cysteines is spatially adjacent to potentially form disulfide linkage. Treatment of Vf FKBP12 with reductant dithiothreitol (DTT) abolished the formation of Vf FKBP12-FK506-CaN ternary complex. Site-directed mutagenesis to substitute one of the cysteines, Cys²⁶, with Ser produced a similar effect as DTT treatment. These results indicate that an intramolecular disulfide bond is a novel structural feature required for the low–affinity interaction between plant FKBP12 and CaN. In conclusion, plant FKBP12 proteins have evolved structural changes that modify their protein-protein interacting domains and cause loss of function against the drug targets.