白消安在斑马鱼早期胚胎和幼鱼发育过程中的毒理和致畸作用

白消安(1,4-二甲磺酸丁酯)是目前在治疗慢性嗜中性粒细胞白血病和骨髓移植中使用最广泛的一种摄生体,因为它在临床上的重要作用,其胚胎毒性长期以来一直是医生们关注的焦点。本研究的目的是评估白消安在斑马鱼胚胎和幼鱼早期发育过程中的毒理作用,包括胚胎毒性和致畸效应。研究首先确定了白消安对斑马鱼胚胎和幼鱼的半致死浓度(LD50),在白消安/DMSO溶液中孵育24h的胚胎其LD50值为43.42μg·mL^-1, 幼鱼的LD50则为237.33μg·mL^-1;其次对白消安处理导致的形态学缺陷也进行了研究,主要包括钩形尾、无尾、脊椎弯曲、脊索波浪状扭曲、单胸鳍、围心包水肿、卵黄囊吸收延迟以及短体长;第三,利用阿利辛蓝(Alcian Blue)染色研究了颌骨和脊椎骨的异常发育,白消安处理过的幼鱼的麦克尔氏软骨(Meckel's cartilage)间的距离显著短于未处理过的阴性对照组,而处理组的幼鱼在脊椎骨的分化程度上也明显低于对照组;另外,为了研究白消安对斑马鱼生殖系统的影响,使用生殖细胞特异性的nanos1 cRNA探针标定斑马鱼原始生殖细胞,研究处理前后原始生殖细胞的增殖和迁移状况,发现暴露在40μg·mL^-1的白消安/DMSO溶液中12h,不仅能够抑制原始细胞的增殖而且明显干扰了这些细胞的定向迁移。这种对细胞的迁移作用之前从未见诸报道,其机制需要进一步地深入研究。     

Identification of a Novel Selenium-containing Compound,Selenoneine, as the Predominant Chemical Form of Organic Selenium in the Blood of Bluefin Tuna

A novel selenium-containing compound having a selenium atom in the imidazole ring, 2-selenyl-N_α,N_α,N_α-trimethyl-l-histidine, 3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate, was identified from the blood and other tissues of the bluefin tuna, Thunnus orientalis. The selenium-containing compound was purified from the tuna blood in several chromatographic steps. High resolution mass spectrometry and nuclear magnetic resonance spectroscopy showed that the exact mass of the [M+H]⁺ ion of the compound was 533.0562 and the molecular formula was C₁₈H₂₉N₆O₄Se₂. Its gross structure was assigned as the oxidized dimeric form of an ergothioneine selenium analog in which the sulfur of ergothioneine is replaced by selenium. Therefore, we named this novel selenium-containing compound “selenoneine.” By speciation analysis of organic selenium compounds using liquid chromatography inductively coupled plasma mass spectrometry, selenoneine was found widely distributed in various tissues of the tuna, with the highest concentration in blood; mackerel blood contained similar levels. Selenoneine was measurable at 2–4 orders of magnitude lower concentration in a limited set of tissues from squid, tilapia, pig, and chicken. Quantitatively, selenoneine is the predominant form of organic selenium in tuna tissues.     

The Nicotine-Evoked Locomotor Response: A Behavioral Paradigm for Toxicity Screening in Zebrafish (Danio rerio) Embryos and Eleutheroembryos Exposed to Methylmercury

This study is an adaptation of the nicotine-evoked locomotor response (NLR) assay, which was originally utilized for phenotype-based neurotoxicity screening in zebrafish embryos. Zebrafish embryos do not exhibit spontaneous swimming until roughly 4 days post-fertilization (dpf), however, a robust swimming response can be induced as early as 36 hours post-fertilization (hpf) by means of acute nicotine exposure (30–240μM). Here, the NLR was tested as a tool for early detection of locomotor phenotypes in 36, 48 and 72 hpf mutant zebrafish embryos of the non-touch-responsive maco strain; this assay successfully discriminated mutant embryos from their non-mutant siblings. Then, methylmercury (MeHg) was used as a proof-of-concept neurotoxicant to test the effectiveness of the NLR assay as a screening tool in toxicology. The locomotor effects of MeHg were evaluated in 6 dpf wild type eleutheroembryos exposed to waterborne MeHg (0, 0.01, 0.03 and 0.1μM). Afterwards, the NLR assay was tested in 48 hpf embryos subjected to the same MeHg exposure regimes. Embryos exposed to 0.01 and 0.03μM of MeHg exhibited significant increases in locomotion in both scenarios. These findings suggest that similar locomotor phenotypes observed in free swimming fish can be detected as early as 48 hpf, when locomotion is induced with nicotine.     

NO VEIN Mediates Auxin-Dependent Specification and Patterning in the Arabidopsis Embryo,Shoot, and Root

Local efflux-dependent auxin gradients and maxima mediate organ and tissue development in plants. Auxin efflux is regulated by dynamic expression and subcellular localization of the PIN auxin-efflux proteins, which appears to be established not only through a self-organizing auxin-mediated polarization mechanism, but also through other means, such as cell fate determination and auxin-independent mechanisms. Here, we show that the Arabidopsis thaliana NO VEIN (NOV) gene, encoding a novel, plant-specific nuclear factor, is required for leaf vascular development, cellular patterning and stem cell maintenance in the root meristem, as well as for cotyledon outgrowth and separation. nov mutations affect many aspects of auxin-dependent development without directly affecting auxin perception. NOV is required for provascular PIN1 expression and region-specific expression of PIN7 in leaf primordia, cell type–specific expression of PIN3, PIN4, and PIN7 in the root, and PIN2 polarity in the root cortex. NOV is specifically expressed in developing embryos, leaf primordia, and shoot and root apical meristems. Our data suggest that NOV function underlies cell fate decisions associated with auxin gradients and maxima, thus establishing cell type–specific PIN expression and polarity. We propose that NOV mediates the acquisition of competence to undergo auxin-dependent coordinated cell specification and patterning, thereby eliciting context-dependent auxin-mediated developmental responses.     

Enantioselectivity in Developmental Toxicity of rac‐metalaxyl and R‐metalaxyl in Zebrafish (Danio rerio) Embryo

Enantioselectivity of chiral pesticides in environmental safety has attracted more and more attention. In this study, we evaluated the enantioselective toxicity of rac‐metalaxyl and R‐metalaxyl to zebrafish (Danio rerio) embryos through various malformations including pericardial edema, yolk sac edema, crooked body, and short tails. The results showed that there were significant differences in toxicity to zebrafish embryos caused by rac‐metalaxyl and R‐metalaxyl, and the LC₅₀s at 96 h are 416.41 (353.91, 499.29) mg · L^‐1 and 320.650 (279.80, 363.46) mg · L^‐1, respectively. In order to explore the possible mechanism of the development defects, the genes involved in the hypothalamic–pituitary–gonadal axis (vtg1, vtg2, cyp17, cyp19a, cyp19b) and hypothalamic–pituitary–thyroid axis (dio1, dio2, nis, tg, tpo) were quantified by quantitative real‐time polymerase chain reaction (qRT‐PCR). The results revealed that there were no significant differences in the expression of vtg1, vtg2, cyp17, cyp19a, and cyp19b after exposure to rac‐metalaxyl. However, the expression of vtg1, cyp19a, and cyp19b decreased significantly after exposure to R‐metalaxyl. And likewise, rac‐metalaxyl only caused the upregulation of dio2, while R‐metalaxyl suppressed the expression of dio1 and tpo and induced the expression of dio2 and nis. The change of gene expression may cause the enantioselectivity in developmental toxicity in zebrafish embryo. The data provided here will be helpful for us to comprehensively understand the potential ecological risks of the currently used chiral fungicides. Chirality 28:489–494, 2016. © 2016 Wiley Periodicals, Inc.     

斑马鱼新基因KLP的筛选、克隆及初步分析

通过重组cDNA表达文库的血清学分析(SEREX)获得来自斑马鱼的cDNA序列,提取斑马鱼总RNA通过RT-PCR得到该基因全长共5171 bp编码1574个氨基酸,运用生物信息学研究工具进行分析显示该基因为一新基因,命名为KLP,在斑马鱼胚胎各个发育时期用RT-PCR检测该基因的表达情况发现:在斑马鱼早期胚胎发育的几个重要时期该基因都有高表达。该基因含有8个锌指结构,其中3个KAISO蛋白特征性的C2H2锌指结构,且此区域在多个物种内高度保守,提示其在胚胎发育早期过程中具有非常重要的作用。     

Mechanisms underlying long- and short-range nodal signaling in Zebrafish

Precise regulation of the signaling range of secreted molecules is essential for proper pattern formation during development. The Nodal family of TGF-beta proteins has been shown to function as both short- and long-range signals. But the underlying mechanisms remain elusive. In this study, we investigated the regulation of the signaling range of zebrafish Nodal proteins Cyclops and Squint, which are short- and long-range signals, respectively. We show that (1) the stability of Cyclops and Squint correlates with the activity range but increasing the stability of the short-range Cyclops does not increase its signaling range; (2) structural differences in the N-terminus region of the mature peptides of Cyclops and Squint determine their differences in the signaling range and swapping the N-terminus region of the Squint mature ligand into that of Cyclops makes the latter function at a distance.     

植物金属硫蛋白及其重金属解毒机制研究进展

金属硫蛋白是一类分子量较小、富含Cys的金属结合蛋白,广泛分布于生物界。近年来从植物中克隆到许多编码金属硫蛋白的基因,并在研究基因表达模式、组织表达特异性以及基因结构,如启动子、内含子在染色体上的定位等方面取得了一定进展,但对其功能的研究还处于起步阶段。很多实验表明,植物金属硫蛋白可以通过其大量的Cys残基螯合重金属并清除活性氧,使植物避免氧化损伤。文章介绍了植物金属硫蛋白的分类、特征、基因结构及其在植物重金属解毒中的作用。      

Distinct Detoxification Mechanisms Confer Resistance to Mesotrione and Atrazine in a Population of Waterhemp

Previous research reported the first case of resistance to mesotrione and other 4-hydroxyphenylpyruvate dioxygenase (HPPD) herbicides in a waterhemp (Amaranthus tuberculatus) population designated MCR (for McLean County mesotrione- and atrazine-resistant). Herein, experiments were conducted to determine if target site or nontarget site mechanisms confer mesotrione resistance in MCR. Additionally, the basis for atrazine resistance was investigated in MCR and an atrazine-resistant but mesotrione-sensitive population (ACR for Adams County mesotrione-sensitive but atrazine-resistant). A standard sensitive population (WCS for Wayne County herbicide-sensitive) was also used for comparison. Mesotrione resistance was not due to an alteration in HPPD sequence, HPPD expression, or reduced herbicide absorption. Metabolism studies using whole plants and excised leaves revealed that the time for 50% of absorbed mesotrione to degrade in MCR was significantly shorter than in ACR and WCS, which correlated with previous phenotypic responses to mesotrione and the quantity of the metabolite 4-hydroxy-mesotrione in excised leaves. The cytochrome P450 monooxygenase inhibitors malathion and tetcyclacis significantly reduced mesotrione metabolism in MCR and corn (Zea mays) excised leaves but not in ACR. Furthermore, malathion increased mesotrione activity in MCR seedlings in greenhouse studies. These results indicate that enhanced oxidative metabolism contributes significantly to mesotrione resistance in MCR. Sequence analysis of atrazine-resistant (MCR and ACR) and atrazine-sensitive (WCS) waterhemp populations detected no differences in the psbA gene. The times for 50% of absorbed atrazine to degrade in corn, MCR, and ACR leaves were shorter than in WCS, and a polar metabolite of atrazine was detected in corn, MCR, and ACR that cochromatographed with a synthetic atrazine-glutathione conjugate. Thus, elevated rates of metabolism via distinct detoxification mechanisms contribute to mesotrione and atrazine resistance within the MCR population.Waterhemp (Amaranthus tuberculatus) is a troublesome annual weed species in midwestern U.S. corn (Zea mays) and soybean (Glycine max) production. The change to production systems with limited tillage has favored waterhemp germination and growth (Hager et al., 2002). Waterhemp seeds are small, and one female plant can produce up to one million seeds (Steckel et al., 2003), which endow waterhemp with an effective short-distance dispersal mechanism. In addition, multiple herbicide resistance mechanisms in waterhemp are facilitated by its dioecious biology and wind-pollinated flowers (Steckel, 2007). The long-distance flow of pollen may be one of the main reasons that multiple herbicide resistance in waterhemp has become widespread in the United States (Liu et al., 2012).Mesotrione (2-[4-(methylsulfonyl)-2-nitrobenzoyl]-1,3-cyclohexanedione) belongs to the triketone class of 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides (Beaudegnies et al., 2009). Molecular information regarding plant HPPD gene sequences and expression patterns is limited (for review, see Pallett, 2000; Kim and Petersen, 2002; Riechers and Stanford, 2002; Matringe et al., 2005), and only a single expressed HPPD gene was detected in waterhemp (Riggins et al., 2010). Herbicidal activity of mesotrione in sensitive plants is due to competitive inhibition of the HPPD enzyme, which is a key enzyme in the biosynthesis of tocopherols and plastoquinone. Plastoquinone is an electron acceptor for the phytoene desaturase reaction in the pathway of carotenoid biosynthesis and also serves as an electron acceptor in PSII (Hess, 2000). Tocopherols and carotenoids are responsible for the detoxification of reactive oxygen species and scavenging of free radicals in plant tissues (Maeda and DellaPenna, 2007; Triantaphylidès and Havaux, 2009; Mène-Saffrané and DellaPenna, 2010), and carotenoids also protect chlorophyll from photooxidation (Cazzonelli and Pogson, 2010). Following mesotrione treatment, carotenoid biosynthesis is inhibited in sensitive plants, resulting in bleaching and necrosis. In particular, new leaves and meristems are primarily affected due to the need for protective carotenoids and tocopherols in photosynthetic tissues (Triantaphylidès and Havaux, 2009) and the systemic nature of mesotrione, which is translocated in the phloem (Mitchell et al., 2001; Beaudegnies et al., 2009).There are two main mechanisms of herbicide resistance in plants: (1) target site alterations, such as mutations that affect herbicide-binding kinetics or amplification of the target site gene (Powles and Yu, 2010), and (2) nontarget site mechanisms, including metabolism, translocation, and sequestration (Yuan et al., 2007; Powles and Yu, 2010). Metabolic detoxification is a common nontarget-based mechanism for herbicide resistance, which typically may result from elevated levels of cytochrome P450 monooxygenase (P450) or glutathione S-transferase (GST) activity (Powles and Yu, 2010; Délye et al., 2011). In addition to conferring resistance in weeds, these enzymes also confer natural tolerance in crops (Kreuz et al., 1996; Riechers et al., 2010). Similar to tolerant sorghum (Sorghum bicolor) lines (Abit and Al-Khatib, 2009), corn is tolerant to mesotrione via rapid metabolism (i.e. ring hydroxylation catalyzed by P450 activity) in combination with slower uptake relative to sensitive weeds and a less sensitive form of the HPPD enzyme in grasses relative to dicots (Hawkes et al., 2001; Mitchell et al., 2001).Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-S-triazine) is a symmetrical triazine herbicide commonly used in corn to selectively control annual dicot weeds. Atrazine disrupts electron transport by competing with plastoquinone for the secondary electron-accepting plastoquinone-binding site on the D1 protein of PSII in chloroplasts (Hess, 2000). Atrazine resistance in weeds can be due to a mutation in the psbA gene that causes a Ser-Gly substitution at amino acid position 264 of the D1 protein (Hirschberg and McIntosh, 1983; Devine and Preston, 2000). Corn and grain sorghum are naturally tolerant to atrazine via the rapid metabolism of atrazine through conjugation with reduced glutathione (GSH; Frear and Swanson, 1970; Lamoureux et al., 1973), which is catalyzed by GST activities (Shimabukuro et al., 1971). Enhanced metabolism of atrazine and simazine in weedy species has been reported in Abutilon theophrasti, Lolium rigidum, and Alopecurus myosuroides due to either GST- or P450-mediated detoxification mechanisms (Burnet et al., 1993; Gray et al., 1996; Cummins et al., 1999; Délye et al., 2011).A population of waterhemp (designated MCR for McLean County mesotrione- and atrazine-resistant) from Illinois is resistant to HPPD inhibitors (Hausman et al., 2011) and atrazine as well as to acetolactate synthase (ALS)-inhibiting herbicides. A different population of waterhemp (designated ACR for Adams County mesotrione-sensitive but atrazine-resistant; Patzoldt et al., 2005) that is atrazine resistant but sensitive to mesotrione (Hausman et al., 2011) and a waterhemp population (designated WCS for Wayne County herbicide-sensitive; Patzoldt et al., 2005) that is sensitive to both mesotrione and atrazine (Hausman et al., 2011) were used in comparison with MCR in this research. MCR displayed 10- and 35-fold resistance to mesotrione in comparison with ACR and WCS, respectively, in greenhouse studies (Hausman et al., 2011). In addition, waterhemp populations with similar patterns of multiple resistance have recently been identified (Hausman et al., 2011; McMullan and Green, 2011; Heap, 2012). However, the mechanisms of resistance to mesotrione and atrazine in these waterhemp populations are currently unknown. Therefore, the objective of this study was to determine if the multiple-herbicide-resistant phenotype of MCR (in regard to mesotrione and atrazine resistance) is due to either target site or nontarget site mechanisms.     

r84, a Novel Therapeutic Antibody against Mouse and Human VEGF with Potent Anti-Tumor Activity and Limited Toxicity Induction

Vascular endothelial growth factor (VEGF) is critical for physiological and pathological angiogenesis. Within the tumor microenvironment, VEGF functions as an endothelial cell survival factor, permeability factor, mitogen, and chemotactic agent. The majority of these functions are mediated by VEGF-induced activation of VEGF receptor 2 (VEGFR2), a high affinity receptor tyrosine kinase expressed by endothelial cells and other cell types in the tumor microenvironment. VEGF can also ligate other cell surface receptors including VEGFR1 and neuropilin-1 and -2. However, the importance of VEGF-induced activation of these receptors in tumorigenesis is still unclear. We report the development and characterization of r84, a fully human monoclonal antibody that binds human and mouse VEGF and selectively blocks VEGF from interacting with VEGFR2 but does not interfere with VEGF∶VEGFR1 interaction. Selective blockade of VEGF binding to VEGFR2 by r84 is shown through ELISA, receptor binding assays, receptor activation assays, and cell-based functional assays. Furthermore, we show that r84 has potent anti-tumor activity and does not alter tissue histology or blood and urine chemistry after chronic high dose therapy in mice. In addition, chronic r84 therapy does not induce elevated blood pressure levels in some models. The ability of r84 to specifically block VEGF∶VEGFR2 binding provides a valuable tool for the characterization of VEGF receptor pathway activation during tumor progression and highlights the utility and safety of selective blockade of VEGF-induced VEGFR2 signaling in tumors.     

EMBRYONIC FLOWER2, a Novel Polycomb Group Protein Homolog, Mediates Shoot Development and Flowering in Arabidopsis

In higher plants, developmental phase changes are regulated by a complex gene network. Loss-of-function mutations in the EMBRYONIC FLOWER genes (EMF1 and EMF2) cause Arabidopsis to flower directly, bypassing vegetative shoot growth. This phenotype suggests that the EMF genes play a major role in repression of the reproductive program. Positional cloning of EMF2 revealed that it encodes a zinc finger protein similar to FERTILIZATION-INDEPENDENT SEED2 and VERNALIZATION2 of Arabidopsis. These genes are characterized as structural homologs of Suppressor of zeste 12 [Su(z)12], a novel Polycomb group gene currently identified in Drosophila. In situ hybridization studies have demonstrated that EMF2 RNA is found in developing embryos, in both the vegetative and the reproductive shoot meristems, and in lateral organ primordia. Transgenic suppression of EMF2 produced a spectrum of early-flowering phenotypes, including emf2 mutant-like phenotype. This result confirms the role of EMF2 in phase transitions by repressing reproductive development.     

三台花中的一新奇化合物--Serratumin A

从云南西双版纳产的三台花（Ｃｌｅｒｏｄｅｎｄｒｕｍ ｓｅｒｒａｔｕｍ ｖａｒ．ａｍｐｌｅｘｉｆｏｌｉｕｍ Ｍｏｌｄｅｎｋｅ）的地上部分分离到６个化合物,它们的结构通过波谱解析（包括２Ｄ ＮＭＲ技术）得到鉴定。其中化合物１是一新奇的单萜烯酸和单糖衍生物的缩合物,命名为ｓｅｒｒａｔｕｍｉｎ Ａ（１）;化合物２～６为首次从该植物中分离得到。     

斑马鱼早期内胚层发育及其分子调控机制

斑马鱼已成为研究脊椎动物胚胎发育的理想模式生物,它具有体外受精、发育周期快、胚体透明等优点,此外其胚胎发育过程中的信号通路与哺乳动物有很高的同源性。该文阐述了斑马鱼早期内胚层发育的过程及其分子调控机制。     

Influence of pH on Ammonia Accumulation and Toxicity in Halophilic, Methylotrophic Methanogens

We studied the effects of pH and ammonia concentration on the growth of three methanogens. These three halophilic, methylotrophic methanogens, Methanolobus bombayensis, Methanolobus taylorii, and Methanohalophilus zhilinaeae, grew at environmental pH ranges that overlapped with each other and spanned the pH range from 7.0 to 9.5. During growth they had reversed membrane pH gradients ((Delta)pH) at all pH values tested. The (Delta)pH was in the range of -0.4 to -0.9 pH units, with the cytosol being more acidic than the environmental pH. Methanohalophilus zhilinaeae had the most negative (Delta)pH (-0.9 pH units). These negative pH gradients resulted in the accumulation of ammonium (NH(inf4)(sup+)), and when grown at the highest external ammonia concentrations that allowed good growth, cells had cytosolic NH(inf4)(sup+) concentrations as high as 180 mM. The high concentrations of cytosolic NH(inf4)(sup+) were accompanied by greater (Delta)pH and lower concentrations of the major cytosolic cation K(sup+) (compared with cells grown in medium with only 5 mM ammonia). Methanolobus bombayensis and Methanolobus taylorii were more sensitive to total external ammonia at higher external pH values, but the inhibitory concentration of un-ionized ammonia that resulted in a 50% reduction of the growth rate was about 2 to 5 mM, regardless of the pH. This is consistent with growth inhibition by ammonia in other bacteria. However, Methanohalophilus zhilinaeae was more resistant to un-ionized ammonia than any other known organism. It had a 50% inhibitory concentration for un-ionized ammonia of 13 mM at pH 8.5 and 45 mM at pH 9.5. We examined the effects of pH on three ammonia-assimilating activities (glutamine synthetase, glutamate dehydrogenase, and alanine dehydrogenase) in cell lysates and found that the pH ranges were consistent with the observed ranges of intracellular pH.     

Carbonic Anhydrase Inhibitors Induce Developmental Toxicity During Zebrafish Embryogenesis,Especially in the Inner Ear

In vertebrates, carbonic anhydrases (CAs) play important roles in ion transport and pH regulation in many organs, including the eyes, kidneys, central nervous system, and inner ear. In aquatic organisms, the enzyme is inhibited by various chemicals present in the environment, such as heavy metals, pesticides, and pharmaceuticals. In this study, the effects of CA inhibitors, i.e., sulfonamides [ethoxyzolamide (EZA), acetazolamide (AZA), and dorzolamide (DZA)], on zebrafish embryogenesis were investigated. In embryos treated with the sulfonamides, abnormal development, such as smaller otoliths, an enlarged heart, an irregular pectoral fin, and aberrant swimming behavior, was observed. Especially, the development of otoliths and locomotor activity was severely affected by all the sulfonamides, and EZA was a consistently stronger inhibitor than AZA or DZA. In the embryos treated with EZA, inner ear hair cells containing several CA isoforms, which provide HCO₃ ^? to the endolymph for otolith calcification and maintain an appropriate pH there, were affected. Acridine orange/ethidium bromide staining indicated that the hair cell damage in the inner ear and pectral fin is due to apoptosis. Moreover, RNA measurement demonstrated that altered gene expression of cell cycle arrest- and apoptosis-related proteins p53, p21, p27, and Bcl-2 occurred even at 0.08 ppm with which normal development was observed. This finding suggests that a low concentration of EZA may affect embryogenesis via the apoptosis pathway. Thus, our findings demonstrated the importance of potential risk assessment of CA inhibition, especially regarding the formation of otoliths as a one of the most sensitive organs in embryogenesis.     

A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development,Aflatoxin Accumulation,and Seed Infection

Caleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein in Aspergillus flavus that is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway, aflR and aflD, were downregulated in the strains in which A. flavus PXG (AfPXG) was silenced, leading to reduced aflatoxin B1 production in vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in which AfPXG was silenced. PXG-deficient A. flavus strains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease.     

CSE1L,a Novel Microvesicle Membrane Protein,Mediates Ras-Triggered Microvesicle Generation and Metastasis of Tumor Cells

Tumor-derived microvesicles are rich in metastasis-related proteases and play a role in the interactions between tumor cells and tumor microenvironment in tumor metastasis. Because shed microvesicles may remain in the extracellular environment around tumor cells, the microvesicle membrane protein may be the potential target for cancer therapy. Here we report that chromosome segregation 1–like (CSE1L) protein is a microvesicle membrane protein and is a potential target for cancer therapy. v-H-Ras expression induced extracellular signal–regulated kinase (ERK)-dependent CSE1L phosphorylation and microvesicle biogenesis in various cancer cells. CSE1L overexpression also triggered microvesicle generation, and CSE1L knockdown diminished v-H-Ras–induced microvesicle generation, matrix metalloproteinase (MMP)-2 and MMP-9 secretion and metastasis of B16F10 melanoma cells. CSE1L was preferentially accumulated in microvesicles and was located in the microvesicle membrane. Furthermore, anti-CSE1L antibody–conjugated quantum dots could target tumors in animal models. Our findings highlight a novel role of Ras-ERK signaling in tumor progression and suggest that CSE1L may be involved in the “early” and “late” metastasis of tumor cells in tumorigenesis. Furthermore, the novel microvesicle membrane protein, CSE1L, may have clinical utility in cancer diagnosis and targeted cancer therapy.