Expression of the Tobacco Ext 1.4 Extensin Gene Upon Mechanical Constraint and Localization of Regulatory Regions

Abstract: The regulation of the Ext 1.4 gene encoding a tobacco ( Nicotiana tabacum L.) extensin was studied in response to mechanical constraints. Transgenic plants carrying chimeric Ext 1.4 promoter/GUS (β-glucuronidase)/ nos terminator or Ext 1.4 3'-end constructs were obtained. Expression of gene fusions was found in tissues where mechanical stresses occur, e.g., during germination, as well as in root and stem tissues. Chimeric genes were successively and transiently expressed in different tissues during germination, i.e., at the tip of the root and then in the hypocotyl, during their growth through the seed coat. Moreover, they were expressed in cortical cells surrounding the emergence of adventitious and lateral roots and developmentally-regulated in nodes. The expression of Ext 1.4 could be induced by imposing mechanical constraints due to curving of either the stems or roots. Expression then occurred in cells where it does not normally occur, i.e., in cortical cells of internodes and in the distal piliferous zone of roots. Accumulation of RNAs occurs several days after the start of the constraint. Promoter regions involved in regulation of expression of Ext 1.4 in stems, roots, and in seedlings upon mechanical constraint could be localized. Moreover, the 3' non-coding region was shown to modulate expression in roots. These results suggest that the regulation of Ext 1.4 following mechanical stress is dependent on both tissue-specific and mechanical-responsive elements.     

Comparing the Environmental Performance of Fluorescent Whitening Agents with Peroxide Bleaching of Mechanical Pulp

The use of fluorescent whitening agents (FWAs) instead of oxidative bleaching agents such as peroxide is an alternative for the bleaching of mechanical pulp. By this approach, the chromophores of the wood components in the pulp are not destroyed chemically but the brightness of the pulp is achieved by increased re-emission of blue light in the range of 400–500 nm. In this study, a typical FWA and peroxide bleaching chemicals are compared with respect to both production and application in the pulp mill. The life-cycle inventory shows that, on the one hand, the production of the FWA leads to higher releases of salts and adsorbable organically bound halogens (AOX) to surface waters and that, on the other hand, significantly less FWA is required in the application step in order to reach the same pulp brightness. The life-cycle impact assessment of the production step is presented in terms of Eco-indicator 95. These results, however, do not cover the environmental fate of various chemicals released to the aquatic environment in the course of the bleaching/whitening step. Therefore, this part is assessed by means of a more detailed investigation of the chemicals' environmental fate in rivers and their aquatic toxicity.     

Role of regulator of G-protein signaling 2 (RGS2) in periodontal ligament cells under mechanical stress

Mechanical stress is thought to regulate the expression of genes in the periodontal ligament (PDL) cells. Using a microarray approach, we recently identified a regulator of G-protein signaling 2 (RGS2) as an up-regulated gene in the PDL cells under compressive force. The RGS protein family is known to turn off G-protein signaling. G-protein signaling involves the production of cAMP, which is thought to be one of the biological mediators in response to mechanical stress. Here, we investigated the role of RGS2 in the PDL cells under mechanical stress. PDL cells derived from the ligament tissues of human premolar teeth were cultured in collagen gels and subjected to static compressive force. Compressive force application time-dependently enhanced RGS2 expression and intracellular cAMP levels. To examine the interrelationship between RGS2 and cAMP, the PDL cells were treated with 2',5'-dideoxyadenosine (DDA), an inhibitor of adenyl cyclase, or antisense S-oligonucleotide (S-ODN) to RGS2 under compressive force. DDA dose-dependently inhibited RGS2 stimulated by compressive force. Blockage of RGS2 by antisense S-ODN elevated the cAMP levels compared with controls. These results indicate that cAMP stimulates RGS2 expression, which in turn leads to a decrease in the cAMP production by inactivating the G-protein signaling in the mechanically stressed PDL cells.     

Separation and quantification of the cellular thiol pool of pea plants treated with heat, salt and atrazine

A novel procedure for the separation of the cellular thiol pool according to the molecular weight and localization of compounds with sulphydryl groups is presented. This simple and rapid method allows the differentiation of thiols into three major fractions-low molecular weight (LMT, primarily glutathione and free cysteine), protein-bound (TPT) and pellet-bound (PBT, associated with cell walls and broken organelles). Moreover, determination of the ratio between surface (readily reactive) thiols (ATG) and those that are more or less buried in the protein structure (BTG) can be achieved. In intact pea leaves, the amounts of the total thiols (LMT+PBT+TPT) varies from 2.5 to 4.8 micromol/g of fresh material. The data for LMT, PBT and TPT were related to each other in the approximate ratio 1:2:7. Treatments of pea plants with high temperature, salinity and low amounts of atrazine affect these sulphydryl types differently. For a greater understanding of the applicability of this method to physiological research, the main mechanisms leading to alterations in the cellular thiol pool are discussed. Furthermore, it is suggested that the proportion of available to buried thiols (ATG/BTG) in proteins could be used as a convenient marker for stress impacts.     

原纤毛－多囊蛋白复合物在肾和骨组织中的类似作用

纤毛－多囊蛋白复合物的功能或者结构异常,是导致常染色体显性多囊肾病的主要原因.该复合物除了被认为在正常的肾上皮细胞上起着机械和化学感受器的作用,可能在骨细胞中也有类似的作用.本文总结了多囊蛋白和纤毛的结构、分布特点以及在肾发育过程中所发挥的作用;着重综述了纤毛 多囊蛋白复合物在肾上皮细胞上作为机械和化学感受器,通过影响细胞内一系列的信号途径,调控细胞的基因转录和蛋白合成的最新研究进展,包括与细胞内钙离子变化有关的钙调神经磷酸酶－NFAT途径和PI3K－Atk途径,调控细胞周期的JAK－STAT途径,及维持正常肾结构的Wnt/β连环蛋白信号途径等;还将通过比较在肾上皮细胞上纤毛 多囊蛋白复合物所激活的信号传导途径和在骨细胞中传导机械刺激的信号转导途径的类同,提示在骨细胞中,纤毛 多囊蛋白复合物可能起着在肾上皮细胞上类似的机械感受器作用,为系统性阐明多囊肾病的发病机制,以及揭示失重或负重状态下骨细胞机械感受的分子机制提供了一个新思路.     

Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum

Background and Aims Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than −180 °C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues.Methods Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1·9 g H₂O g⁻¹ dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 °C s⁻¹ or programmed cooling at 3·3 °C s⁻¹. Samples were thawed rapidly (177 °C s⁻¹) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination.Key Results Immediately following thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem, which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the possibility for, the extent of and the vigour of regrowth.Conclusions Autophagic degradation and ultimately autolysis of cells following cryo-exposure and formation of small (0·2–0·4 µm) intracellular ice crystals challenges current ideas that ice causes immediate physical damage to cells. Instead, freezing stress may induce a signal for programmed cell death (PCD). Cells that form more ice crystals during cooling have faster PCD responses.     

丛枝菌根真菌对刺槐幼苗机械损伤响应机制的初步研究

通过对刺槐幼苗每隔3d剪去1片叶片造成持续机械损伤,测定了0~138h抗氧化酶活性变化及刺槐幼苗生长情况;同时使用孔径25μm尼龙网设置三室根箱隔网系统,测定了供体瞬时机械损伤后受体的抗氧化酶活性的持续变化,探讨接种丛枝菌根真菌刺槐幼苗对持续及瞬时机械损伤后的响应机制,以及菌根菌丝桥对刺槐幼苗机械损伤信号的传递特征。结果表明:在持续机械损伤胁迫下,接种丛枝菌根真菌能够促进刺槐幼苗的根系生长、提高幼苗的成活率,接种丛枝菌根真菌的刺槐幼苗成活率及根系鲜重比对照分别增加15.38%和23.52%。瞬时机械损伤后0、48、90、114、138h刺槐幼苗的苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)活性都呈现先增加后降低的趋势,均在90h达到最大值,并且菌根化幼苗的PAL和POD活性显著高于未菌根化幼苗。瞬时机械损伤后,菌根菌丝桥能够介导刺槐幼苗间相关信号的传递,从而引起菌根化受体刺槐幼苗的PAL和POD活性表现出与供体机械损伤幼苗相同的变化趋势。     

Biomimetic repeat protein derived from Xenopus tropicalis for fibrous scaffold fabrication

Collagen, silk, and elastin are the fibrous proteins consist of representative amino acid repeats. Because these proteins exhibited distinguishing mechanical properties, they have been utilized in diverse applications, such as fiber‐based sensors, filtration membranes, supporting materials, and tissue engineering scaffolds. Despite their infinite prevalence and potential, most studies have only focused on a few repeat proteins. In this work, the hypothetical protein with a repeat motif derived from the frog Xenopus tropicalis was obtained and characterized for its potential as a novel protein‐based material. The codon‐optimized recombinant frog repeat protein, referred to as ‘xetro’, was produced at a high rate in a bacterial system, and an acid extraction‐based purified xetro protein was successfully fabricated into microfibers and nanofibers using wet spinning and electrospinning, respectively. Specifically, the wet‐spun xetro microfibers demonstrated about 2‐ and 1.5‐fold higher tensile strength compared with synthetic polymer polylactic acid and cross‐linked collagen, respectively. In addition, the wet‐spun xetro microfibers showed about sevenfold greater stiffness than collagen. Therefore, the mass production potential and greater mechanical properties of the xetro fiber may result in these fibers becoming a new promising fiber‐based material for biomedical engineering. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 659–664, 2015.