Double Strand Breaks and Cell-Cycle Arrest Induced by the Cyanobacterial Toxin Cylindrospermopsin in HepG2 Cells

The newly emerging cyanobacterial cytotoxin cylindrospermopsin (CYN) is increasingly found in surface freshwaters, worldwide. It poses a potential threat to humans after chronic exposure as it was shown to be genotoxic in a range of test systems and is potentially carcinogenic. However, the mechanisms of CYN toxicity and genotoxicity are not well understood. In the present study CYN induced formation of DNA double strand breaks (DSBs), after prolonged exposure (72 h), in human hepatoma cells, HepG2. CYN (0.1–0.5 µg/mL, 24–96 h) induced morphological changes and reduced cell viability in a dose and time dependent manner. No significant increase in lactate dehydrogenase (LDH) leakage could be observed after CYN exposure, indicating that the reduction in cell number was due to decreased cell proliferation and not due to cytotoxicity. This was confirmed by imunocytochemical analysis of the cell-proliferation marker Ki67. Analysis of the cell-cycle using flow-cytometry showed that CYN has an impact on the cell cycle, indicating G0/G1 arrest after 24 h and S-phase arrest after longer exposure (72 and 96 h). Our results provide new evidence that CYN is a direct acting genotoxin, causing DSBs, and these facts need to be considered in the human health risk assessment.     

基于iTRAQ技术的干旱胁迫下玉米苗期蛋白质组学研究

采用同位素标记相对定量(iTRAQ)技术,对干旱胁迫条件下苗期玉米的蛋白质组学变化进行分析。结果表明,共检测到玉米幼苗中的207个蛋白在干旱胁迫后发生了显著的丰度变化。根据蛋白注释情况可将这些蛋白归入信号传导、渗透调节、蛋白合成与折叠、ROS清除、膜运输、转录相关、细胞结构与细胞周期、脂肪酸代谢、碳水化合物与能量代谢、光合作用与光呼吸等代谢途径。干旱胁迫后,涉及光反应和呼气作用的差异蛋白多表现为丰度上升;涉及碳水化合物及蛋白质合成差异蛋白多表现为丰度下降;与渗透调节相关的脱水蛋白、脯氨酸代谢和渗透胁迫相关的蛋白酶则显示为丰度上升。干旱胁迫还能导致植物体内活性氧大量产生,活性氧清除相关的酶类也会发生明显的丰度上升。根据研究结果推测,玉米苗期主要通过降低植株生长速率、减少水分散失、清除自由基等多种方式维持其在干旱胁迫条件下的生长发育过程。     

Migration of gluten under shear flow as a novel mechanism for separating wheat flour into gluten and starch

This paper describes a novel principle for the separation of wheat flour into starch and gluten in a concentrated medium. The process is based on the use of simple shear flow in a cone-and-cone device. The separation takes place in two steps. Initially, local segregation of gluten and starch phases occurs, leading to formation of macroscopically visible gluten patches distributed throughout the dough. This local segregation can be understood by considering the dough as a visco-elastic matrix containing an inert filler (starch). Further shearing leads to aggregation of those patches and migration (large-scale separation) towards the apex of the cone. As a result, the wheat dough is separated into a protein-poor fraction, containing less than 4% protein, and a protein-rich fraction containing almost 50% protein on a dry weight basis. However, under the process conditions used, upon a very long shearing, a redistribution of the aggregated gluten structures in the starch phase was observed, demonstrating a processing limit for the separation performance. Compared to traditional processing, the separation process presented shows opportunities for producing high quality gluten accompanied with significant water savings. Considering the fact that simple shear flow in steady rate is less harmful to gluten quality, such a separation process could benefit gluten quality.     

Microstructure formation and rheological behaviour of dough under simple shear flow

In a z-blade mixer, both shear and extensional deformations contribute to the development of dough structure. I effect of simple shearing versus z-blade mixing at similar levels of work input, on the microstructure and uniaxial extensional properties of two doughs prepared from flours of different strengths. With respect to microstructure, mixing initially increased the formation of coarse protein patches, leading to a heterogeneous dough structure with a high fracture stress (σ_max) and significant strain hardening. These parameters decreased with prolonged mixing. This was accompanied by loss of glutenin macro polymer (GMP) wet weight and formation of a more homogenous microstructure. Prolonged mixing typically led to an over-mixed state. In contrast, prolonged simple shearing did not affect GMP content or strain hardening and gave enhanced shear banding. Confocal scanning laser microscopy revealed that short-term simple shearing induced structure formation in the direction of the shear flow for both flour types, followed by formation of shear-banded gluten structures both parallel and perpendicular to the direction of shear flow. Uniaxial extension of dough oriented parallel or perpendicular to the shear field did not reveal anisotropy. Apparently, the observed heterogeneity on a scale of ‘mm’ was not relevant for this type of rheology. Nevertheless, a relative weakening of dough strength (reduced fracture stress) was observed as a function of long-term shearing. This seems to be related to a local segregation effect caused by differences in visco-elasticity between the gluten phase and the starch granules. The results of this study reveal important features of the dough processing and underline the importance of not only work input, but also the type of deformation applied.     

水稻小粒基因SG101的鉴定和精细定位

【目的】籽粒大小是决定水稻产量的重要农艺性状之一,开展水稻籽粒大小相关基因的克隆和功能研究对于阐述水稻产量形成的遗传调控机制具有重要意义。【方法】利用甲基磺酸乙酯诱变粳稻品种中花11,筛选获得一小粒突变体,命名为sg101(small grain 101)。通过形态学、细胞学手段调查了SG101的突变对籽粒大小、穗部主要性状及颖壳细胞数目和大小的影响,通过测定叶夹角和胚芽鞘长度分析其对外施油菜素内酯的差异响应,结合定量PCR技术分析了油菜素内酯合成途径和信号途径相关基因表达情况,并利用图位克隆的手段精细定位了水稻小粒基因SG101。【结果】与野生型相比,突变体sg101粒长和粒宽均极显著减小,从而导致千粒重极显著降低。此外,sg101还表现出结实率降低、穗长变短、二次枝梗数减少、植株变矮等。细胞学观察发现sg101的颖壳细胞大小没有改变,但细胞数目明显减少。定量PCR检测表明sg101中的细胞周期相关基因表达显著下降。另外,突变体sg101对外施油菜素内酯响应迟钝,其油菜素内酯合成途径和信号途径相关基因表达亦显著降低。【结论】遗传分析表明sg101突变体由隐性单基因控制,通过图位克隆的方法将SG101精细定位于第1染色体上,物理距离为265 kb的区间内。这为该基因的克隆及深入的功能研究奠定了基础。     

磷素水平对小麦突变体农艺性状和品质特性的影响

为丰富磷高效小麦遗传资源,分析了小麦纤维素相关基因突变体（ZC5和ZC7,为郑麦9023经EMS诱导的脆杆小麦）及其野生型（小麦品种郑麦9023）在三种磷素水平下（0、105、210 kg·hm^-2）、不同发育时期的相关生理指标和成熟期农艺和品质性状。结果表明,基因型、磷水平和基因型与磷水平互作均对旗叶叶面积、旗叶SPAD值和籽粒淀粉含量有一定影响,影响程度因生育时期而异。不同处理小麦灌浆过程符合Logistic生长规律,籽粒干物质积累均呈“慢－快－慢”的变化趋势。基因型和磷水平均对成熟期籽粒蛋白质含量和全磷含量有极显著影响;基因型对成熟期株高、有效小穗数、穗粒数及千粒重有极显著影响。相关分析表明,籽粒渐增期灌浆速率和干物质积累量、快增期干物质积累量和旗叶SPAD值均与千粒重呈极显著正相关,籽粒渐增期、快增期持续时间和淀粉含量均与千粒重呈显著正相关。ZC5为磷敏感材料,施磷能提前ZC5最大灌浆速率出现日期,提高其最大灌浆速率和平均灌浆速率,且随着施磷量的增加籽粒蛋白质和全磷含量增加。纤维素合成相关基因突变对旗叶SPAD值、叶面积、籽粒淀粉含量、蛋白质含量、全磷含量、株高、有效小穗数、穗粒数和千粒重均存在显著影响。通过化学诱变改良小麦细胞壁成分可为小麦育种提供丰富的遗传资源。     

Trafficking and deposition of prolamins in wheat

The processing properties of wheat flour are largely determined by the structures and interactions of the grain storage protein, the so called “gluten proteins”. The synthesis, folding and deposition of the gluten proteins take place within the endomembrane system of the plant cell. Several studies have suggested that two trafficking pathways occur in wheat, with the proteins either being transported via the Golgi apparatus into the vacuole or assembling within the ER to form protein bodies that are subsequently internalized into vacuoles by a process analogous to autophagy. The precise routes of trafficking and deposition of individual gluten proteins remain unclear and so are the molecular and cellular mechanisms regulating their sorting in the two pathways.     

Distinct functions of <Emphasis Type="Italic">S. pombe</Emphasis>Rec12 (Spo11) protein and Rec12-dependent crossover recombination (chiasmata) in meiosis I; and a requirement for Rec12 in meiosis II

BACKGROUND: In most organisms proper reductional chromosome segregation during meiosis I is strongly correlated with the presence of crossover recombination structures (chiasmata); recombination deficient mutants lack crossovers and suffer meiosis I nondisjunction. We report that these functions are separable in the fission yeast Schizosaccharomyces pombe. RESULTS: Intron mapping and expression studies confirmed that Rec12 is a member of the Spo11/Top6A topoisomerase family required for the formation of meiotic dsDNA breaks and recombination. rec12-117, rec12-D15 (null), and rec12-Y98F (active site) mutants lacked most crossover recombination and chromosomes segregated abnormally to generate aneuploid meiotic products. Since S. pombe contains only three chromosome pairs, many of those aneuploid products were viable. The types of aberrant chromosome segregation were inferred from the inheritance patterns of centromere linked markers in diploid meiotic products. The rec12-117 and rec12-D15 mutants manifest segregation errors during both meiosis I and meiosis II. Remarkably, the rec12-Y98F (active site) mutant exhibited essentially normal meiosis I segregation patterns, but still exhibited meiosis II segregation errors. CONCLUSIONS: Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I. Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I. In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II.     

Modulation of fibroblast inflammatory response by surface modification of a perfluorinated ionomer

An ideal surface for implantable glucose sensors would be able to evade the events leading to chronic inflammation and fibrosis, thereby extending its utility in an in vivo environment. Nafion?, a perfluorinated ionomer, is the membrane material preferred for in situ glucose sensors. Unfortunately, the surface properties of Nafion? promote random protein adsorption and eventual foreign body encapsulation, thus leading to loss of glucose signal over time. Details of the techniques to render Nafion? nonprotein fouling are given in a previous article [T. I. Valdes et al., Biomaterials 29, 1356 (2008)]. Once random protein adsorption is prevented, a biologically active peptide can be covalently bonded to the treated Nafion? to induce cellular adhesion. Cellular responses to these novel decorated Nafion? surfaces are detailed here, including cell viability, cell spreading, and type I collagen synthesis. Normal human dermal fibroblasts (NHDFs) were cultured on control and modified Nafion? surfaces. Findings indicate that Nafion? modified with 10% 2-hydroxyethyl methacrylate and 90% tetraglyme created a nonfouling surface that was subsequently decorated with the YRGDS peptide. NHDFs were shown to have exhibited decreased type I collagen production in comparison to NHDF cells on unmodified Nafion? surfaces. Here, the authors report evidence that proves that optimizing conditions to prevent protein adsorption and enhance cellular adhesion may eliminate fibrous encapsulation of an implant.     

Effects of Cylindrospermopsin Producing Cyanobacterium and Its Crude Extracts on a Benthic Green Alga—Competition or Allelopathy?

Cylindrospermopsin (CYN) is a toxic secondary metabolite produced by filamentous cyanobacteria which could work as an allelopathic substance, although its ecological role in cyanobacterial-algal assemblages is mostly unclear. The competition between the CYN-producing cyanobacterium Chrysosporum (Aphanizomenon) ovalisporum, and the benthic green alga Chlorococcum sp. was investigated in mixed cultures, and the effects of CYN-containing cyanobacterial crude extract on Chlorococcum sp. were tested by treatments with crude extracts containing total cell debris, and with cell debris free crude extracts, modelling the collapse of a cyanobacterial water bloom. The growth inhibition of Chlorococcum sp. increased with the increasing ratio of the cyanobacterium in mixed cultures (inhibition ranged from 26% to 87% compared to control). Interestingly, inhibition of the cyanobacterium growth also occurred in mixed cultures, and it was more pronounced than it was expected. The inhibitory effects of cyanobacterial crude extracts on Chlorococcum cultures were concentration-dependent. The presence of C. ovalisporum in mixed cultures did not cause significant differences in nutrient content compared to Chlorococcum control culture, so the growth inhibition of the green alga could be linked to the presence of CYN and/or other bioactive compounds.     

Identification of Dynamic Changes in Proteins Associated with the Cellular Cytoskeleton after Exposure to Okadaic Acid

Exposure of cells to the diarrhetic shellfish poison, okadaic acid, leads to a dramatic reorganization of cytoskeletal architecture and loss of cell-cell contact. When cells are exposed to high concentrations of okadaic acid (100–500 nM), the morphological rearrangement is followed by apoptotic cell death. Okadaic acid inhibits the broad acting Ser/Thr protein phosphatases 1 and 2A, which results in hyperphosphorylation of a large number of proteins. Some of these hyperphosphorylated proteins are most likely key players in the reorganization of the cell morphology induced by okadaic acid. We wanted to identify these phosphoproteins and searched for them in the cellular lipid rafts, which have been found to contain proteins that regulate cytoskeletal dynamics and cell adhesion. By using stable isotope labeling by amino acids in cell culture cells treated with okadaic acid (400 nM) could be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton, microtubules and cell adhesion structures. A large number of these okadaic acid-regulated proteins have previously also been shown to be similarly regulated prior to cell proliferation and migration. Our results suggest that okadaic acid activates general cell signaling pathways that induce breakdown of the cortical actin cytoskeleton and cell detachment.     

转录组测序分析甘蓝型油菜DW871矮化性状

DW871是一个具有特异株型的甘蓝型油菜矮化品系.为了研究DW871的相关基因及其表达模式,我们以甘蓝型高秆油菜HW871为对照,选取抽薹期茎秆组织进行转录组基因表达分析.结果显示:DW871与HW871之间存在8665个显著差异基因,其中上调基因2582个,下调基因6083个.通过GO富集分析可注释到3个大类共22个...     

The use of genetics in understanding protein composition and grain quality in wheat

As each of the classes of wheat-grain protein has been implicated in some aspect of man's interest in wheat utilization, we are motivated to learn more about the genetic control of their synthesis so that breeders may better tailor wheats to specific requirements. The gliadins have particularly merited study because they appear to be responsible for coeliac condition and for depressed lysine content, and as they are proving valuable for varietal identification and grain-quality prediction. Genetic aspects of gliadin synthesis have been studied using aneuploids, by examining F₁ and F₂ segregation after crossing, and by computer comparison of the gliadin composition of many genotypes in conjunction with systematic pedigree comparisons. These studies indicate gliadin synthesis to be controlled by blocks of tightly linked genes on the short arms of the chromosomes of groups 1 and 6. The high molecular weight subunits of glutenin are genetically distinct from the gliadins, being coded by genes on the long arms of chromosomes 1A, 1B and 1D. A better understanding of the relationship between grain quality and specific endosperm proteins is now developing. It is likely to provide simpler means of selecting for quality type in both breeding and harvest segregation.     

Chromatidenspaltung beim Merkmal Nematodenresistenz der Kartoffel

Zusammenfassung Aus Untersuchungen zur Vererbung der monogen dominant bedingten Resistenzgegen Pathotyp 1 (=ROI) des KartoffelnematodenGlobodera rostochiensis ergaben sich Hinweise für Chromatidenspaltung. Bei der Schaffung von multiplexen Vererbern der Resistenz sind zahlreiche Testkreuzungen und Resistenzuntersuchungen von Nachkommen notwendig. Für die Ermittlung des Umfangs der Testpopulationen und ihre genetische Zuordnung wurde eine grafische Darstellung erarbeitet.

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Summary Potato clones which are multiplex for monogene dominant resistance toGlobodera rostochiensis must be test-crossed to susceptible partners to determine their genotypes. The following allowances were calculated for the limits of the populations of seedlings to be tested α=5% (risk group 1), \=5% (risk group 2);d=10% (least significant difference). The following values were obtained for chromosome segregation: triplex/quadruplex 35, duplex 215, simplex 320 and nulliplex 35. For chromatid segregation the values were: quadruplex 35, triplex 80, duplex 248, simplex 320 and nulliplex 35. For practical reasons the 95% confidence limits were displayed graphically. From it the following numbers of seedlings to be tested were calculated: between quadruplex/triplex and duplex, 38 for chromosome segregation and 56 for chromatid segregation respectively; between duplex and simplex, 32 for chromosome segregation and 38 for chromatid segregation respectively. Statistical testing of the segregation ratios withX ² and t-test gave for the triplex and duplex cases highly significant deviations from the expected values for chromosome segregation and a good fit for the chromatid segregation.

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Résumé Les clones de pommes de terre présentant une résistance monogénique dominante à hérédité multiple pourGlobodera rostochiensis, ne peuvent être reconnus qu'après hybridation avec des clones sensibles. Le nombre de plantules à tester dans une population a été calculé selon les critères suivants: α=5% (risque de le ordre), β=5% (risque de 2e ordre).d=10% (différence minimale à la probabilité constante de l'hypothèse nulle, considérée comme valable pour la pratique). Les valeurs suivantes ont été obtenues pour la division des chromosomes: triplex/quadruplex 35, duplex 215, simplex 320 et nulliplex 35. Les valeurs pour la division des chromatides sont: quadruplex 35, triplex 80, duplex 248, simplex 320 et nulliplex 35. Pour l'usage pratique, les intervalles de confiance à 95% ont été présentés graphiquement. Les valeurs suivants ont été obtenues pour le nombre minimum de plantules à tester: entre quadruplex/triplex et duplex 38 pour la division des chromosomes et 56 pour celle des chromatides, entre duplex et simplex 32 lorsqu'il s'agit de division des chromosomes et 38 pour celle des chromatides. L'examen statistique des proportions de ségrégation par les testsX ² et t ont donné des écarts hautement significatifs des valeurs théoriques pour les cas de triplex et duplex, lors des divisions des chromosomes, et des valeurs comparables aux espérances, lors des divisions de chromatides.

     

MG-132, an inhibitor of proteasomes and calpains,induced inhibition of oocyte maturation and aneuploidy in mouse oocytes

BACKGROUND: Although chromosome missegregation during oocyte maturation (OM) is a significant contributor to human morbidity and mortality, very little is known about the causes and mechanisms of aneuploidy. Several investigators have proposed that temporal perturbations during OM predispose oocytes to aberrant chromosome segregation. One approach for testing this proposal is to temporarily inhibit the activity of protein proteolysis during OM. We used the reversible proteasome inhibitor MG-132 to transiently perturb the temporal sequence of events during OM and subsequently analyzed mouse metaphase II (MII) for cytogenetic abnormalities. The transient inhibition of proteasome activity by MG-132 resulted in elevated levels of oocytes containing extra chromatids and chromosomes. RESULTS: The transient inhibition of proteasome-mediated proteolysis during OM by MG-132 resulted in dose-response delays during OM and elevated levels of aneuploid MII oocytes. Oocytes exposed in vitro to MG-132 exhibited greater delays during metaphase I (MI) as demonstrated by significantly (p < 0.01) higher levels of MI arrested oocytes and lower frequencies of premature sister chromatid separation in MII oocytes. Furthermore, the proportions of MII oocytes containing single chromatids and extra chromosomes significantly (p < 0.01) increased with MG-132 dosage. CONCLUSIONS: These data suggest that the MG-132-induced transient delay of proteasomal activity during mouse OM in vitro predisposed oocytes to abnormal chromosome segregation. Although these findings support a relationship between disturbed proteasomal activity and chromosome segregation, considerable additional data are needed to further investigate the roles of proteasome-mediated proteolysis and other potential molecular mechanisms on chromosome segregation during OM.     

植物表观遗传修饰的分子机制及其生物学功能

表观遗传调控是指基于非DNA序列的改变所致基因功能的变化,最终导致生物多效性表型。表观遗传调控因子通过多种途径调控基因表达,参与植物几乎所有的生长发育过程。本文介绍了DNA甲基化、组蛋白修饰、RNA甲基化、染色质重塑、非编码RNA、基因印记和母性效应等植物表观遗传修饰主要类型的分子机制,以及它们在细胞增殖、细胞分化、生物与非生物胁迫、开花、果实成熟和胚胎发育中的作用机制和生物学功能,并对其在农业上的应用作了展望。     

Resistance to potato virus Y inSolanum tuberosum spp.andigena

Tests were made on lines ofSolanum tuberosum spp.andigena to determine 1) whether resistance to potato virus Y exists in the Andigena germplasm, 2) the nature of its inheritance, and 3) the type of resistance. Results of the isolation and identification studies indicated that the pathogen involved was a common strain of potato virus Y. Under field conditions susceptible plants frequently escaped infection. However, field exposures over two seasons resulted in the same ratios as tests in which the same progenies were mechanically inoculated. Mechanical inoculation at the seedling stage proved to be a reliable means of transmission and resulted in accurate screening for resistance. Of 641 tub × tub clones tested, all were susceptible to the virus. Of 366 tub × adg clones tested, 170 were resistant and 196 were susceptible. This fits a 13:15 ratio, assuming random chromatid segregation and a single dominant gene conferring resistance. Plants that were resistant following mechanical transmission were also resistant when inoculated by aphids, indicating the reliability of mechanical transmission as a means of screening for resistance. To determine the type of resistance, top-graft and approach-graft tests were made. Failure to recover the virus from resistant plants inoculated by either grafting method suggests that immunity is the type of resistance involved.     

玉米自交系齐319空间诱变系的生物学效应分析

以两份玉米子粒类型空间诱变系SP-M1、SP-M2以及野生型玉米自交系齐319为试验材料,对田间农艺性状、穗部性状、子粒的营养品质性状和胚乳内部显微结构进行分析。结果表明,与野生型相比,两个诱变系在株高、穗位高、粒长、百粒重等田间农艺性状和穗部性状上发生显著或极显著变异,且变异方向不同,SP-M1和SP-M2赖氨酸含量显著高于野生型,SP-M2淀粉含量显著高于野生型,蛋白质含量显著低于野生型。2个诱变系与齐319在胚乳边缘组织、胚乳内部组织、淀粉粒、基质蛋白等结构存在差异,淀粉粒的组成比例和排列顺序是影响胚乳超微结构差异的主要原因。     

Palmitic Acid and Ergosta-7,22-dien-3-ol Contribute to the Apoptotic Effect and Cell Cycle Arrest of an Extract from Marthasterias glacialis L. in Neuroblastoma Cells

We describe the effect of a chemically characterized lipophilic extract obtained from Marthasterias glacialis L. against human breast cancer (MCF-7) and human neuroblastoma (SH-SY5Y) cell lines. Evaluation of DNA synthesis revealed that both cell lines were markedly affected in a concentration-dependent way, the SH-SY5Y cell line being more susceptible. Cell cycle arrest was observed, an effect induced by the sterol, ergosta-7,22-dien-3-ol, present in the extract. Morphological evaluation of treated cells showed the advent of lipid droplets and chromatin condensation compatible with apoptosis, which was confirmed by the evaluation of caspase-3 and -9 activities. Palmitic acid was the main compound responsible for this apoptotic effect by a ceramide-independent mechanism that involved endoplasmic reticulum (ER)-stress with upregulation of CCAAT/-enhancer-binding protein homologous protein (CHOP).