Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes

NPR1 is an essential regulator of plant systemic acquired resistance (SAR), which confers immunity to a broad-spectrum of pathogens. SAR induction results in accumulation of the signal molecule salicylic acid (SA), which induces defense gene expression via activation of NPR1. We found that in an uninduced state, NPR1 is present as an oligomer formed through intermolecular disulfide bonds. Upon SAR induction, a biphasic change in cellular reduction potential occurs, resulting in reduction of NPR1 to a monomeric form. Monomeric NPR1 accumulates in the nucleus and activates gene expression. Inhibition of NPR1 reduction prevents defense gene expression, whereas mutation of Cys82 or Cys216 in NPR1 leads to constitutive monomerization, nuclear localization of the mutant proteins, and defense gene expression. These data provide a missing link between accumulation of SA and activation of NPR1 in the SAR signaling pathway.     

Comparison of Endoscopic Submuscosal Implantation vs. Surgical Intramuscular Implantation of VX2 Fragments for Establishing a Rabbit Esophageal Tumor Model for Mimicking Human Esophageal Squamous Carcinoma

Purpose

This study was undertaken to establish a rabbit esophageal tumor model for mimicking human esophageal squamous carcinoma (ESC) by endoscopic and surgical implantation of VX2 tumors.

Methods

Fragments of a VX2 tumour were endoscopically implanted in the submucosal layer of the thoracic esophagus of 32 New Zealand white rabbits, while 34 animals received surgical implantation into the muscular layer. Then, the animals were studied endoscopically and pathologically. The safety and efficiency of the two methods and the pathological features of the animal models were analyzed.

Results

Both the endoscopic and the surgical method had a relatively high success rate of tumor implantation [93.7% (30/32) vs. 97.1% (33/34)] and tumor growth [86.7% (26/30) vs. 81.8% (27/33)], and the variation in the results was not statistically significant (P>0.05). Compared with those produced by the surgical method, the models produced by the endoscopic method had a higher rate of severe esophageal stricture [61.5% (16/26) vs. 29.6% (8/27)] and of intra-luminal tumor growth [73.1% (19/26) vs. 37.0% (10/27)], and had a lower rate of tumor invasion of adjacent organs [53.8% (14/26) vs. 81.5% (22/27)]; all of these results were statistically significant (P<0.05). However, the difference in the survival time and the rates of tumor regional/distant metastasis [38.5% (10/26) vs. 51.8% (14/27)] between the two methods were not statistically significant (P>0.05).

Conclusion

The endoscopic and surgical methods are both safe and effective for establishment of VX2 tumors in the rabbit esophagus. The models produced by the two methods have different pathologic features mimicking that of human ESC. We recommend the models for studies on surgical procedures and minimally invasive treatments.     

Polysaccharides from Lycium barbarum leaves: Isolation, characterization and splenocyte proliferation activity

The polysaccharides from the fruits of Lycium barbarum have received considerable attention in previous publication, but the polysaccharides from the leaves were rarely reported. In the present work, four water-soluble polysaccharide fractions: LBP-I, LBP-II, LBP-III and LBP-IV isolated from L. barbarum leaves were purified through DEAE-Sephadex A-25. LBP-II and LBP-IV respectively showed one symmetrical peak on HPGPC with average molecular weight of 9.39×10(4)Da and 4.18×10(5)Da. UV and IR analysis of the two fractions showed the characteristics of acidic polysaccharides combined with polypeptides or proteins. GC analysis showed LBP-IV was mainly composed of rhamnose, arabinose, xylose, glucose and galactose with molar ratio of 1.61:3.82:3.44:7.54:1.00, and the uronic acid content was 47.68% (w/w) determined by sulfuric acid-carbazole method. (1)H and (13)C NMR spectra of LBP-IV also showed the presence of carboxyl carbon and five anomeric carbons, and suggested there may be both α- and β-anomeric configurations in this fraction. Moreover, splenocyte proliferation activity assay showed that LBP-IV significantly enhanced the proliferation of splenocyte stimulated by ConA or LPS, indicating the fraction has the beneficial effect on immunostimulating activity.     

离子对高效液相色谱法分析黄瓜花叶病毒侵染烟草的总RNA水平变化

为研究植物病毒在RNA水平上对寄主植物的基因表达产生的影响,采用离子对高效液相色谱法,对黄瓜花叶病毒侵染烟草造成的总RNA的组成成分变化进行研究。离子对液相色谱法,是近几年才应用于对RNA进行分离、纯化和分析的一种试验技术,具有操作简单、重复性好、所需时间短等优点。选择适宜的离子对试剂,并对选定的离子对试剂正己胺/1,1,1,3,3,3-六氟-2-丙醇进行了优化,达到了较好的总RNA分离效果,并观察到健康植株和染病植株分离峰之间的差异,有差异的RNA种类还需进一步试验来验证。为研究植物病毒的致病机制提供一种新的试验方法和途径。     

Steady‐state and time‐resolved fluorescence of tetramethylrhodamine attached to DNA: correlation with DNA sequences

Time‐resolved fluorescence as well as steady‐state absorption and fluorescence were detected in order to study the interactions between tetramethylrhodamine (TAMRA) and DNA when TAMRA was covalently labeled on single‐ and double‐stranded oligonucleotides. Fluorescence intensity quenching and lifetime changes were characterized and correlated with different DNA sequences. The results demonstrated that the photoinduced electron transfer interaction between guanosine residues and TAMRA introduced a short lifetime fluorescence component when guanosine residues were at the TAMRA‐attached terminal of the DNA sequences. The discrepancy of two‐state and three‐state models in previous studies was due to the DNA sequence selection and sensitivity of techniques used to detect the short lifetime component. The results will help the design of fluorescence‐based experiments related to a dye labeled probe. Copyright © 2012 John Wiley & Sons, Ltd.     

Anti-osteopontin monoclonal antibody prevents ovariectomy-induced osteoporosis in mice by promotion of osteoclast apoptosis

Osteopontin (OPN) is abundant in mineralized tissues and has long been implicated in bone remodeling. However, the therapeutic effect of targeting OPN in bone loss diseases and the underlying molecular mechanism remain largely unknown. Here, we reported that anti-OPN mAb (23C3) could protect against ovariectomy-induced osteoporosis in mice, demonstrated by microcomputed tomography analysis and histopathology evaluation. In vitro assay showed that 23C3 mAb reduced osteoclasts (OCs)-mediated bone resorption through promotion of mature OC apoptosis. Thus, the study has important implications for understanding the role of OPN in OC bone resorption and survival, and OPN antagonists may have therapeutic potential for osteoporosis and other osteopenic diseases.     

Upregulation of TrkB Promotes Epithelial-Mesenchymal Transition and Anoikis Resistance in Endometrial Carcinoma

Mechanisms governing the metastasis of endometrial carcinoma (EC) are poorly defined. Recent data support a role for the cell surface receptor tyrosine kinase TrkB in the progression of several human tumors. Here we present evidence for a direct role of TrkB in human EC. Immunohistochemical analysis revealed that TrkB and its secreted ligand, brain-derived neurotrophic factor (BDNF), are more highly expressed in EC than in normal endometrium. High TrkB levels correlated with lymph node metastasis (p<0.05) and lymphovascular space involvement (p<0.05) in EC. Depletion of TrkB by stable shRNA-mediated knockdown decreased the migratory and invasive capacity of cancer cell lines in vitro and resulted in anoikis in suspended cells. Conversely, exogenous expression of TrkB increased cell migration and invasion and promoted anoikis resistance in suspension culture. Furthermore, over-expression of TrkB or stimulation by BDNF resulted in altered the expression of molecular mediators of the epithelial-to-mesenchymal transition (EMT). RNA interference (RNAi)-mediated depletion of the downstream regulator, Twist, blocked TrkB-induced EMT-like transformation. The use of in vivo models revealed decreased peritoneal dissemination in TrkB-depleted EC cells. Additionally, TrkB-depleted EC cells underwent mesenchymal-to-epithelial transition and anoikis in vivo. Our data support a novel function for TrkB in promoting EMT and resistance to anoikis. Thus, TrkB may constitute a potential therapeutic target in human EC.     

Regulation of ciliary beat frequency by the nitric oxide signaling pathway in mouse nasal and tracheal epithelial cells

Objectives

Our purpose was to investigate the role of the nitric oxide (NO) signaling pathway in the regulation of ciliary beat frequency (CBF) in mouse nasal and tracheal epithelial cells.

Methods

We studied the effects of the NO donor l-arginine (L-Arg) and specific inhibitors of the NO signaling pathway on CBF of both nasal and tracheal epithelial cells by using high-speed digital microscopy. We also examined eNOS, sGC β, PKG I and acetylated α tubulin expression in native mouse nasal and tracheal epithelium using immunohistochemical methods.

Results

L-Arg significantly increased CBF of cultured nasal and tracheal epithelial cells, and the effects were blocked by pretreatment with N^G-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, with LY-83583, a sGC inhibitor, or with KT-5823, a PKG inhibitor. Positive immunostaining for NO signaling molecules including eNOS, sGC β and PKG I was observed in either nasal or tracheal ciliated epithelium.

Conclusion

NO plays a role in regulating CBF of mouse respiratory epithelial cells via a eNOS–NO–sGC β–cGMP–PKG I pathway.     

热带假丝酵母脂肪醛脱氢酶基因CtAld1和CtAld2的功能评价

【目的】热带假丝酵母是发酵法生产二元酸的重要工业菌株,具有较高的ω-氧化活性。脂肪醛脱氢酶在ω-氧化途径中起重要作用,催化脂肪醛生成脂肪酸,但其具体催化功能及对细胞生理影响还未被系统研究。本文通过删除脂肪醛脱氢酶基因CtAld1和CtAld2鉴定了其在ω-氧化途径中的功能。【方法】通过基因组信息挖掘获得热带假丝酵母脂肪醛脱氢酶基因CtAld1和CtAld2序列,在此基础上,通过同源重组敲除CtAld1和CtAld2基因。考察突变株的生长和胞内脂肪醛脱氢酶活性变化,并评价CtAld1和CtAld2基因敲除对细胞二元酸合成能力的影响。【结果】分别获得了热带假丝酵母突变株XZX-1(ΔCtAld1/ΔCtAld1)、XZX-2(ΔCtAld2/ΔCtAld2)和XZX-12(ΔCtAld1/ΔCtAld1,ΔCtAld2/ΔCtAld2)。在以十二烷为唯一碳源的培养基中,敲除CtAld2基因显著抑制细胞的生长,胞内脂肪醛脱氢酶活性降低为出发菌株的30%;敲除CtAld1基因尽管会使细胞损失一部分醛脱氢酶活性,但能够一定程度地提升细胞在十二烷中的生长性能。敲除CtAld1或CtAld2会降低菌株二元酸产量,组合敲除CtAld1和CtAld2严重削弱菌株十二碳二元酸的合成能力。【结论】CtAld2对热带假丝酵母细胞的生长和十二碳二元酸的合成具有重要作用,缺失CtAld1或CtAld2基因降低细胞的二元酸合成能力。CtAld1和CtAld2可作为热带假丝酵母ω-氧化途径代谢工程改造的潜在靶点。