Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a

SaPIN2a, a proteinase inhibitor II from American black nightshade (Solanum americanum Mill.) is highly expressed in the phloem and could be involved in regulating proteolysis in the sieve elements. To further investigate the physiological role of SaPIN2a, we have produced transgenic lettuce (Lactuca sativa L.) expressing SaPIN2a from the CaMV35S promoter by Agrobacterium-mediated transformation. Stable integration of the SaPIN2a cDNA and its inheritance in transgenic lines were confirmed by Southern blot analysis and segregation analysis of the R₁ progeny. SaPIN2a mRNA was detected in both the R₀ and R₁ transformants on northern blot analysis but the SaPIN2a protein was not detected on western blot analysis using anti-peptide antibodies against SaPIN2a. Despite an absence of significant inhibitory activity against bovine trypsin and chymotrypsin in extracts of transgenic lettuce, the endogenous trypsin-like activity in each transgenic line was almost completely inhibited, and the endogenous chymotrypsin-like activity moderately inhibited. Our finding that heterogeneously expressed SaPIN2a in transgenic lettuce inhibits plant endogenous protease activity further indicates that SaPIN2a regulates proteolysis, and could be potentially exploited for the protection of foreign protein production in transgenic plants.Abbreviations CaMV cauliflower mosaic virus - cDNA complementary DNA - NOS nopaline synthase - PAGE polyacrylamide gel electrophoresis - PI proteinase inhibitor - SaPIN2a Solanum americanum proteinase inhibitor IIa - SDS sodium dodecyl sulphate - T-DNA transferred DNA     

微生境对黄梅秤锤树野生种群叶片功能性状的影响

植物功能性状可以反映植物应对环境变化的适应策略。本文以黄梅秤锤树(Sinojackia huangmeiensis)当前唯一野生种群为对象, 比较了3种微生境(湖边、种群中心、耕地边)中该物种的叶片功能性状均值、种内变异和叶片生态化学计量特征的差异, 分析了黄梅秤锤树对湖岸带微生境变化的响应及其适应策略。结果表明: (1) 3种微生境中土壤C、N、P含量没有显著性差异(P > 0.05), 但土壤C∶N和C∶P具有显著性差异(P < 0.05), 土壤类型和养分条件有所不同。(2)黄梅秤锤树叶片功能性状的比较用单因素方差分析和贝叶斯方差分析得出的结果一致, 均为叶长、叶面积和比叶面积在中心区域显著高于湖边(P < 0.05), 而耕地边与湖边和中心区域均没有显著差异(P > 0.05); 叶N含量在湖边显著高于中心区域和耕地边(P < 0.05), 而中心区域和耕地边间没有显著差异(P > 0.05); 叶宽、叶长/叶宽、叶干物质含量、叶C和叶P含量在3种微生境间都没有显著性差异(P > 0.05)。(3)黄梅秤锤树叶片的N∶P在湖边显著高于中心区域和耕地边(P < 0.05), C∶N在湖边显著小于中心区域和耕地边(P < 0.05), N∶P和C∶N在中心区域和耕地边没有显著性差异(P > 0.05), C∶P在3种微生境间都没有显著性差异(P > 0.05)。(4)黄梅秤锤树叶片功能性状的总体变异程度在0.02-0.28之间, 其中叶片C和N含量在湖边和中心区域的种内变异程度显著较低, 表明3种生境中湖边和中心区域黄梅秤锤树种群的稳定性相对较差。(5)湖边黄梅秤锤树主要通过增加叶N含量促进生长; 中心区域黄梅秤锤树主要通过增加叶面积和比叶面积以及提高叶N的利用效率来提高光捕获能力促进生长; 耕地边黄梅秤锤树的叶N含量和叶面积、比叶面积都处于中等水平, 通过性状之间的共同作用使植株生长达到最佳水平。以上结果表明, 由于微地形、水位波动和土壤环境条件的差异, 黄梅秤锤树对3种生境中的适应策略有所不同, 并且不是通过单一性状调整来适应环境的变化, 而是通过多种性状之间的权衡达到更好的适应效果。     

Assessment of Microwave Cooking on the Bioaccessibility of Cadmium from Various Food Matrices Using an In Vitro Digestion Model

Bioaccessibility represents the maximum amount of pollutant ingested with food that is available for intestinal absorption. The measurement of bioaccessibility can achieve a more accurate risk assessment. Thus, in this study, the bioaccessibility of raw/microwave-cooked store-bought food including carrot, potato, white radish, lotus root, sweet corn, long grain rice, soybean, fleshy prawn, eastern oyster, kelp, and common carp were investigated by applying an in vitro digestion method. A validated microwave digestion/ICP-MS method was applied for determining the concentration of Cd. In this study, the concentration of Cd ranged 3.7–215.8 μg/kg fw in which carrot contained the lowest Cd while the fleshy prawn contained the highest Cd. There are no statistical differences of Cd content in microwave-cooked food and raw food except potato, lotus root, and eastern oyster. Cd in most of the cooked food materials was less bioaccessible than in raw food except sweet corn, potato, and kelp. The bioaccessibility of Cd was around 100 % in either raw or cooked potatoes. Microwave cooking caused the decreasing of bioaccessibility around 0–68 %, depending on different food matrix. Maximal decreasing of Cd bioaccessibility occurred in common carp. Thus, microwave cooking could be a feasible strategy for decreasing Cd bioaccessibility. In addition, the Cd dissolution in oral, gastric, and small intestine phase was different in different food matrix. For most of the investigated food items, Cd was largely migrated either into the oral phase (carrot, potato, white radish, lotus root, raw soybean, kelp, and common carp) or into the gastric phase (sweet corn, cooked soybean, rice, fleshy prawn, and eastern oyster). Our findings will have significant implications for food processing aiming to decrease the absorption of Cd and risk assessment analysis improvements. Further study is needed to use the animal model to validate these results.     

Nucleation Promoting Factors Regulate the Expression and Localization of Arp2/3 Complex during Meiosis of Mouse Oocytes

The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs) JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division.     

Evolutionary mode and functional divergence of vertebrate NMDA receptor subunit 2 genes

Background

Ionotropic glutamate receptors in the central nervous system play a major role in numerous brain functions including learning and memory in many vertebrate species. NR2 subunits have been regarded as rate-limiting molecules in controlling the optimal N-methyl-D-aspartate (NMDA) receptor''s coincidence-detection property and subsequent learning and memory function across multi-species. However, its evolutionary mode among vertebrate species remains unclear.

Results

With extensive analysis of phylogeny, exon structure, protein domain, paralogon and synteny, we demonstrated that two-round genome duplication generated quartet GRIN2 genes and the third-round fish-specific genome duplication generated extra copies of fish GRIN2 genes. In addition, in-depth investigation has enabled the identification of three novel genes, GRIN2C_Gg, GRIN2D-1_Ol and GRIN2D-2_Tr in the chicken, medaka and fugu genome, respectively. Furthermore, we showed functional divergence of NR2 genes mostly occurred at the first-round duplication, amino acid residues located at the N-terminal Lig_chan domain were responsible for type I functional divergence between these GRIN2 subfamilies and purifying selection has been the prominent natural pressure operating on these diversified GRIN2 genes.

Conclusion and Significance

These findings provide intriguing subjects for testing the 2R and 3R hypothesis and we expect it could provide new insights into the underlying evolution mechanisms of cognition in vertebrate.     

The fluted giant clam (Tridacna squamosa) increases nitrate absorption and upregulates the expression of a homolog of SIALIN (H+:2NO3− cotransporter) in the ctenidium during light exposure

Coral Reefs - Giant clams flourish in nutrient-poor waters of tropical Indo-Pacific because they live in symbiosis with extracellular dinoflagellates (zooxanthellae) and receive photosynthates from...     

Nogo/RTN4 isoforms and RTN3 expression protect SH-SY5Y cells against multiple death insults

Among the members of the reticulon (RTN) family, Nogo-A/RTN4A, a prominent myelin-associated neurite growth inhibitory protein, and RTN3 are highly expressed in neurons. However, neuronal cell-autonomous functions of Nogo-A, as well as other members of the RTN family, are unclear. We show here that SH-SY5Y neuroblastoma cells stably over-expressing either two of the three major isoforms of Nogo/RTN4 (Nogo-A and Nogo-B) or a major isoform of RTN3 were protected against cell death induced by a battery of apoptosis-inducing agents (including serum deprivation, staurosporine, etoposide, and H₂O₂) compared to vector-transfected control cells. Nogo-A, -B, and RTN3 are particularly effective in terms of protection against H₂O₂-induced increase in intracellular reactive oxygen species levels and ensuing apoptotic and autophagic cell death. Expression of these RTNs upregulated basal levels of Bax, activated Bax, and activated caspase 3, but did not exhibit an enhanced ER stress response. The protective effect of RTNs is also not dependent on classical survival-promoting signaling pathways such as Akt and Erk kinase pathways. Neuron-enriched Nogo-A/Rtn4A and RTN3 may, therefore, exert a protective effect on neuronal cells against death stimuli, and elevation of their levels during injury may have a cell-autonomous survival-promoting function.