The response of <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> CH34 to spaceflight in the international space station

The survival and behavior of Cupriavidus metallidurans strain CH34 were tested in space. In three spaceflight experiments, during three separate visits to the ‘International Space Station’ (ISS), strain CH34 was grown for 10–12 days at ambient temperature on mineral agar medium. Space- and earth-grown cells were compared post-flight by flow cytometry and using 2D-gel protein analysis. Pre-, in- and post-flight incubation conditions and experiment design had a significant impact on the survival and growth of CH34 in space. In the CH34 cells returning from spaceflight, 16 proteins were identified which were present in higher concentration in cells developed in spaceflight conditions. These proteins were involved in a specific response of CH34 to carbon limitation and oxidative stress, and included an acetone carboxylase subunit, fructose biphosphate aldolase, a DNA protection during starvation protein, chaperone protein, universal stress protein, and alkyl hydroperoxide reductase. The reproducible observation of the over-expression of these same proteins in multiple flight experiments, indicated that the CH34 cells could experience a substrate limitation and oxidative stress in spaceflight where cells and substrates are exposed to lower levels of gravity and higher doses of ionizing radiation. Bacterium C. metallidurans CH34 was able to grow normally under spaceflight conditions with very minor to no effects on cell physiology, but nevertheless specifically altered the expression of a few proteins in response to the environmental changes.     

Silencing of the <Emphasis Type="Italic">CCNB1, Her2</Emphasis>, and <Emphasis Type="Italic">PKC</Emphasis> genes by small interfering RNA differently retards the division of different human cancer cell lines

Deregulation of genes encoding proteins responsible for cell cycle control frequently accompanies cell malignization and switches the cell program from differentiation and apoptosis to uncontrollable proliferation. We used siRNAs targeted to HER2, protein kinase C (PKC), and cyclin B1 (CCNB1) mRNAs to evaluate the therapeutic potential of the suppression of genes coding for key cell cycle regulators in different human cancer cells. The CCNB1, HER2, or PKC mRNA levels were efficiently reduced within 48 h after transfection with siCycB1, siHER2 or siPKC, respectively. Silencing of HER2, PKC, and CCNB1 substantially reduced the growth rates of all cell lines under study except HL-60 but did not affect cell death or apoptosis. The most pronounced inhibition of cell division was induced by siCycB1 in SK-N-MC cells and by siPKC in MCF-7 cells. We conclude that the selected siRNAs inhibit tumor cell division, and the investigated genes can be promising targets in cancer treatment.     

Microarray analysis of spaceflown murine thymus tissue reveals changes in gene expression regulating stress and glucocorticoid receptors

The detrimental effects of spaceflight and simulated microgravity on the immune system have been extensively documented. We report here microarray gene expression analysis, in concert with quantitative RT‐PCR, in young adult C57BL/6NTac mice at 8 weeks of age after exposure to spaceflight aboard the space shuttle (STS‐118) for a period of 13 days. Upon conclusion of the mission, thymus lobes were extracted from space flown mice (FLT) as well as age‐ and sex‐matched ground control mice similarly housed in animal enclosure modules (AEM). mRNA was extracted and an automated array analysis for gene expression was performed. Examination of the microarray data revealed 970 individual probes that had a 1.5‐fold or greater change. When these data were averaged (n = 4), we identified 12 genes that were significantly up‐ or down‐regulated by at least 1.5‐fold after spaceflight (P ≤ 0.05). The genes that significantly differed from the AEM controls and that were also confirmed via QRT‐PCR were as follows: Rbm3 (up‐regulated) and Hsph110, Hsp90aa1, Cxcl10, Stip1, Fkbp4 (down‐regulated). QRT‐PCR confirmed the microarray results and demonstrated additional gene expression alteration in other T cell related genes, including: Ctla‐4, IFN‐α2a (up‐regulated) and CD44 (down‐regulated). Together, these data demonstrate that spaceflight induces significant changes in the thymic mRNA expression of genes that regulate stress, glucocorticoid receptor metabolism, and T cell signaling activity. These data explain, in part, the reported systemic compromise of the immune system after exposure to the microgravity of space. J. Cell. Biochem. 110: 372–381, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of salt and osmotic stresses on free polyamine content and expression of polyamine biosynthetic genes in <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Grape (Vitis vinifera L.) seedlings grown in vitro were treated with either 200 mM NaCl or 350 mM mannitol for 7 d. Both salinity and osmotic stress caused significant increase in electrolyte leakage. From the three commonly occurring free polyamines (PA), only conspicuous accumulation of putrescine was found in the NaCl-treated seedlings. Four PA biosynthetic genes encoding arginine decarboxylase (pVvADC), S-adenosylmethionine decarboxylase (pVvSAMDC), spermidine synthase (pVvSPDS) and spermine synthase (pVvSPMS) were successfully isolated. While induction of pVvADC was observed from the 1^st day of salt treatment, pVvSAMDC and pVvSPMS were induced only at late stage of stress. As for expression levels of genes in the mannitol-treated seedling, either temporary (pVvADC at day 1) or late (pVvSPMS at days 5 and 7) induction was observed.     

模拟微重力下秀丽隐杆线虫的力学感知和肌萎缩的发生机制——太空飞行线虫试验地面平行对照研究部分

目前,微重力导致肌萎缩的分子机制尚不清楚,重力感知是该事件发生的关键环节．为了回答这一问题,在此之前首先实施了太空线虫试验,这部分结果已经在本刊报道过．而本次研究主要是在地面上建立了模拟微重力环境,观察处理后秀丽隐杆线虫（C.elegans）体壁肌细胞结构和功能的变化,一方面用于验证太空试验,同时比较两种处理结果的异同,以便于评价地面模拟微重力的有效性．经过14天19．5h旋转模拟微重力处理后,对线虫生存率和运动能力进行了观察,并检测了几个重要的肌相关基因表达和蛋白质水平．模拟微重力下线虫生存率没有明显变化,但运动频率显著下降,爬行轨迹也发生了轻微改变,运动幅度降低,提示线虫运动功能出现障碍．从形态学上观察发现：肌球蛋白A（myosin A）免疫荧光染色显示模拟微重力组肌纤维面积缩小,而肌细胞致密体（dense-body）染色可见荧光亮度下降．这些结果直接提示模拟微重力使线虫出现了肌萎缩．随后Western blotting试验结果揭示,模拟微重力组线虫体壁肌的主要结构蛋白——myosin A含量减少,进一步确证了微重力性肌萎缩发生．在基因水平,旋转后抗肌萎缩蛋白基因（dys-1）表达明显上升,而hlh-1,unc-54,myo-3和egl-19的mRNA水平均下调,提示dys-1在骨骼肌感知和传导力学信息方面有重要作用,而hlh-1,unc-54,myo-3和egl-19则分别从结构和功能两个途径促进了微重力性肌萎缩的发生和发展．本次试验所得到的结果同太空飞行试验结果十分相似,一方面强化了太空试验结论,另一方面说明在地面上模拟微重力对生物体进行研究是有效可行的,将有助于提高太空试验的质量．     

Discovering genes responsible for silk synthesis in Bombyx mori by piggyBac‐based random insertional mutagenesis

Silkworm mutants are valuable resources for both transgenic breeding and gene discovery. PiggyBac-based random insertional mutagenesis has been widely used in gene functional studies. In order to discover genes involved in silk synthesis, a piggyBac-based random insertional library was constructed using Bombyx mori, and the mutants with abnormal cocoon were particularly screened. By this means, a “thin cocoon” mutant was identified. This mutant revealed thinner cocoon shell and shorter posterior silk gland (PSG) compared with the wild type. The messenger RNA (mRNA) levels of all the three fibroin genes, including Fib-H, Fib-L and P25, were significantly down-regulated in the PSG of mutants. Four piggyBac insertion sites were identified in Aquaporin (AQP), Longitudinals lacking protein-like {Lola), Glutamyl aminopeptidase-like (GluAP) and Loc101744460. The mRNA levels of all the four genes were significantly altered in the silk gland of mutants. In particular, the mRNA amount of AQP, a gene responsible for the regulation of osmotic pressure, decreased dramatically immediately prior to the spinning stage in the anterior silk gland of mutants. The identification of the genes disrupted in the “thin cocoon” mutant in this study provided useful information for understanding silk production and transgenic breeding of silkworms in the future.     

Selection of Stable Reference Genes for Quantitative Real-Time PCR on Herbaceous Peony (<i>Paeonia lactiflora</i> Pall.) in Response to Drought Stress

Herbaceous peony (Paeonia lactiflora Pall.), as a high-end cut flower in the international market, has high ornamental and medicinal values. But in Northern China, drought is a major environmental factor influencing the growth and development of P. lactiflora. Quantitative real-time polymerase chain reaction (qRT-PCR) can evaluate gene expression levels under different stress conditions, and stable internal reference is the key for qRT-PCR. At present, there is no systematic screening of internal reference for correcting gene expressions of P. lactiflora in response to drought stress. In this study, 10 candidate genes [ubiquitin (UBQ2), UBQ1, elongation factor 1-α (EF-1α), Histidine (His), eukaryotic initiation factor (eIF), tubulin (TUB), actin (ACT), UBQ3, ACT2, RNA polymerase II (RNA Pol II)] were chosen, and 4 analysis methods were used to compare the stabilities for these 10 genes coping with drought stress. Due to the difference of operation methods, the results of different analysis were distinct, and the final comprehensive analysis indicated that EF-1α was a relatively stable internal reference gene for P. lactiflora under drought stress. Also, UBQ1 and UBQ2 were the best reference gene combination according to GeNorm analysis. This study will lay a foundation for screening the key genes of P. lactiflora in response to drought stress.     

Time Course of <Emphasis Type="Italic">c-fos</Emphasis>, <Emphasis Type="Italic">vasopressin</Emphasis> and <Emphasis Type="Italic">oxytocin</Emphasis> mRNA Expression in the Hypothalamus Following Long-Term Dehydration

Summary 1. This study presents a time course analysis of the messenger RNA (mRNA) levels of c-fos, vasopressin (VP), and oxytocin (OT) in the paraventricular (PVN) and supraoptic nucleus (SON), following acute and chronic dehydration by water deprivation. 2. Male Wistar rats were separated into five groups: nondehydrated (control group) and dehydrated for 6, 24, 48 and 72 h. Following water deprivation, animals were decapitated, their blood was collected for hematocrit, osmolality, and plasma sodium measurements, and brains were removed for dissection of both PVN and SON. 3. As expected, the hematocrit, osmolality, plasma sodium, and weight loss were increased after water deprivation. In SON, a significant increase in both VP and OT mRNA expression was observed 6 h after dehydration reaching a peak at 24 h and returning to basal levels of expression at 72 h. In the PVN, an increase in both VP and OT mRNA expression occurred 24 h after dehydration. At 72 h the VP and OT mRNA expression levels had decreased but they were still at higher levels than those detected in control animals. 4. These results suggest that SON is the first nucleus to respond to the dehydration stimulus. Additionally, we also observed an increase in c-fos mRNA expression in both PVN and SON 6 h after water deprivation, which progressively decreased 24, 48, and 72 h after the onset of water deprivation. Therefore, it is possible that c-fos may be involved in the modulation of VP and OT genes, regulating the mRNA expression levels on a temporally distinct basis within the PVN and SON.     

<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> overexpressing γ-ECS exhibits biotic stress tolerance likely through NPR1-dependent salicylic acid-mediated pathway

The elaborate networks and the crosstalk of established signaling molecules like salicylic acid (SA), jasmonic acid (JA), ethylene (ET), abscisic acid (ABA), reactive oxygen species (ROS) and glutathione (GSH) play key role in plant defense response. To obtain further insight into the mechanism through which GSH is involved in this crosstalk to mitigate biotic stress, transgenic Nicotiana tabacum overexpressing Lycopersicon esculentum gamma-glutamylcysteine synthetase (LeECS) gene (NtGB lines) were generated with enhanced level of GSH in comparison with wild-type plants exhibiting resistance to pathogenesis as well. The expression levels of non-expressor of pathogenesis-related genes 1 (NPR1)-dependent genes like pathogenesis-related gene 1 (NtPR1), mitogen-activated protein kinase kinase (NtMAPKK), glutamine synthetase (NtGLS) were significantly enhanced alongwith NtNPR1. However, the expression levels of NPR1-independent genes like NtPR2, NtPR5 and short-chain dehydrogenase/reductase family protein (NtSDRLP) were either insignificant or were downregulated. Additionally, increase in expression of thioredoxin (NtTRXh), S-nitrosoglutathione reductase 1 (NtGSNOR1) and suppression of isochorismate synthase 1 (NtICS1) was noted. Comprehensive analysis of GSH-fed tobacco BY2 cell line in a time-dependent manner reciprocated the in planta results. Better tolerance of NtGB lines against biotrophic Pseudomonas syringae pv. tabaci was noted as compared to necrotrophic Alternaria alternata. Through two-dimensional gel electrophoresis (2-DE) and image analysis, 48 differentially expressed spots were identified and through identification as well as functional categorization, ten proteins were found to be SA-related. Collectively, our results suggest GSH to be a member in cross-communication with other signaling molecules in mitigating biotic stress likely through NPR1-dependent SA-mediated pathway.     

Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C _q) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (β-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.     

Characterization and phylogenetic analysis of environmental stress-responsive <Emphasis Type="Italic">SAP</Emphasis> gene family encoding A20/AN1 zinc finger proteins in tomato

Characterization of genes responsive to stress is important for efforts on improving stress tolerance of plants. To address components involved in stress tolerance of tomato (Solanum lycopersicum), a stress-responsive gene family encoding A20/AN1 zinc finger proteins was characterized. In the present study, 13 members of this gene family were cloned from tomato cultivar Pusa Ruby and named as Stress Associated Protein (SAP) genes. Out of 13 genes, 12 have been mapped on their respective chromosomes. Expression of these genes in response to cold, heat, salt, desiccation, wounding, abscisic acid, oxidative and submergence stresses was analysed. All tomato SAP genes were found to be responsive to one or other type of environmental stress. The phylogenetic analysis of these genes, along with their orthologs from Solanaceae species suggests the presence of a common set of SAP genes in the studied Solanaceae species. The present study characterizes a SAP gene family, which encodes A20/AN1 zinc finger containing proteins from tomato for the first time. Genes showing high expression in response to a particular stress can be exploited for improving stress tolerance of tomato and other Solanaceae members. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Low temperature promotes intron retention in two <Emphasis Type="Italic">e-cor</Emphasis> genes of durum wheat

Following the screening of a suppression subtractive library developed from durum wheat plants exposed to low temperature for 6 h, two early cold-regulated (e-cor) genes have been isolated. These genes, coding putatively for a ribokinase (7H8) and a C3H2C3 RING-finger protein (6G2), were characterized by the stress-induced retention of a subset of introns in the mature mRNA. This feature was dependent on cold for 7H8 and on cold and dehydration for 6G2. When other genes, such as the stress-related gene WCOR410c, coding for a dehydrin (one intron), or a gene coding for a putative ATP binding cassette transporter (16 introns) were analyzed, no cold-dependent intron retention was observed. Cold-induced intron retention was not observed in mutants defective in the chloroplast development; nevertheless treatment with cycloheximide in the absence of cold was able to promote intron retention for the 7H8 e-cor gene. These results suggest that the cold-induced intron retention reflects the response of the spliceosoma to specific environmental signals transduced to the splicing protein factors through a chloroplast-dependent pathway. Notably, when the 7H8 Arabidopsis orthologous gene was analyzed, no stress induction in terms of mRNA abundance and no cold-dependent intron retention was detected. Otherwise, 6G2 Arabidopsis homologous sequences sharing the same genomic structure of the durum wheat 6G2 showed a similar intron retention event although not strictly dependent on stress.Electronic Supplementary Material Supplementary material is available for this article at     

Molecular characterization of the <Emphasis Type="Italic">Arginine decarboxylase</Emphasis> gene family in rice

Arginine decarboxylase (ADC) is a key enzyme in plants that converts arginine into putrescine, an important mediator of abiotic stress tolerance. Adc genes have been isolated from a number of dicotyledonous plants but the oat and rice Adc genes are the only representatives of monocotyledonous species described thus far. Rice has a small family of Adc genes, and OsAdc1 expression has been shown to fluctuate under drought and chilling stress. We identified and characterized a second rice Adc gene (OsAdc2) which encodes a 629-amino-acid protein with a predicted molecular mass of 67 kDa. An unusual feature of the OsAdc2 gene is the presence of an intron and a short upstream open reading frame in the 5′-UTR. Sequence comparisons showed that OsAdc2 is more closely related to the oat Adc gene than to OsAdc1 or to its dicot homologs, and mRNA analysis showed that the two rice genes are also differently regulated. Whereas OsAdc1 is expressed in leaf, root and stem, OsAdc2 expression is restricted to stem tissue. Protein expression was investigated with specific antibodies against ADC1 and ADC2, corroborating the mRNA data. We discuss the expression profiles of OsAdc1 and OsAdc2 and potential functions for the two corresponding proteins.