The power of statistical tests using field trial count data of nontarget organisms in environmental risk assessment of genetically modified plants

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Cell death via mitochondrial apoptotic pathway due to activation of Bax by lysosomal photodamage

Lysosomal photosensitizers have been used in photodynamic therapy. The combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. Lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, N-aspartyl chlorin e6 (NPe6), an effective photosensitizer that preferentially accumulates in lysosomes, was used to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) after NPe6-photodynamic treatment (NPe6-PDT) was studied using real-time single-cell analysis. Our results demonstrated that NPe6-PDT induced rapid generation of reactive oxygen species (ROS). The photodynamically produced ROS caused a rapid destruction of lysosomes, leading to release of cathepsins, and the ROS scavengers vitamin C and NAC prevent the effects. Then the following spatiotemporal sequence of cellular events was observed during cell apoptosis: Bcl-2-associated X protein (Bax) activation, cytochrome c release, and caspase-9/-3 activation. Importantly, the activation of Bax proved to be a crucial event in this apoptotic machinery, because suppressing the endogenous Bax using siRNA could significantly inhibit cytochrome c release and caspase-9/-3 activation and protect the cell from death. In conclusion, this study demonstrates that PDT with lysosomal photosensitizer induces Bax activation and subsequently initiates the mitochondrial apoptotic pathway.     

季节性流感裂解疫苗在小鼠中针对同型与异型流感病毒的免疫保护效力

为了研究季节性流感裂解疫苗在小鼠中针对甲型流感病毒同型同株、同型异株、异型异株攻击的免疫保护效力及其与诱发的血凝抑制(HI)抗体滴度的关系,本研究使用我国2008~2009年度季节性流感裂解疫苗中不同剂量的甲1型流感病毒H1N1(疫苗株病毒A/Brisbane/59/2007(H1N1)-like)和甲3型流感病毒的H3N2(疫苗株病毒A/Brisbane/10/2007(H3N2)-like)疫苗组分免疫BALB/c小鼠,首先确定了能在小鼠中诱发血HI抗体滴度达到40的疫苗免疫剂量;然后以此剂量免疫小鼠,分别使用同型同株流感病毒(鼠肺适应株A/Brisbane/59/2007(H1N1)-like virus(MA))(简称A1)和同型异株流感病毒(鼠肺适应株A/Purto Rico/8/34(H1N1))(简称PR8)攻击H1N1疫苗免疫小鼠,使用异型异株流感病毒A1攻击H3N2疫苗免疫小鼠,通过体重变化和存活率情况,探讨季节性流感疫苗在小鼠中针对甲型流感病毒同型同株、同型异株、异型异株攻击的保护效力。结果显示,季节性流感裂解疫苗H1N1和H3N2组分按照HA不同剂量0.15μg、0.5μg、1.5μg、5μg和15μg免疫小鼠后,所诱发的HI抗体滴度随免疫剂量的增加而增强,1.5μgHA即可以诱发免疫小鼠HI抗体滴度达到40;以此剂量免疫小鼠,分别使用3LD50、10LD50、30LD50、100LD50、300LD50、1 000LD50和3 000LD50的同型同株流感病毒A1进行攻击,1.5μgH1N1疫苗可以100%保护小鼠抵御高至1000LD50同型同株流感病毒A1的攻击,15μg甚至可以100%保护3 000LD50同型同株流感病毒A1的攻击,但是这两个剂量免疫的小鼠在低至3LD50同型异株流感病毒PR8的攻击后都全部死亡;使用可以诱发HI抗体滴度达到140的15μg H3N2疫苗免疫小鼠,在低至3LD50异型异株流感病毒A1的攻击后亦全部死亡。以上结果表明,季节性流感疫苗可使小鼠HI抗体滴度达到40的疫苗免疫剂量为1.5μg,该免疫剂量可以有效保护小鼠抵御同型同株流感病毒的攻击,但是难以保护小鼠抵御同型异株与异型异株流感病毒的攻击,这一结果为建立以季节性流感疫苗为参考的免疫保护评价体系提供了实验依据。     

Silencing synaptic communication between random interneurons during Drosophila larval locomotion

Genetic manipulation of individual neurons provides a powerful approach toward understanding their contribution to stereotypic behaviors. We describe and evaluate a method for identifying candidate interneurons and associated neuropile compartments that mediate Drosophila larval locomotion. We created Drosophila larvae that express green fluorescent protein (GFP) and a shibire(ts1) (shi(ts1)) transgene (a temperature-sensitive neuronal silencer) in small numbers of randomly selected cholinergic neurons. These larvae were screened for aberrant behavior at an elevated temperature (31-32°C). Among larvae with abnormal locomotion or sensory-motor responses, some had very small numbers of GFP-labeled temperature-sensitive interneurons. Labeled ascending interneurons projecting from the abdominal ganglia to specific brain neuropile compartments emerged as candidates for mediation of larval locomotion. Random targeting of small sets of neurons for functional evaluation, together with anatomical mapping of their processes, provides a tool for identifying the regions of the central nervous system that are required for normal locomotion. We discuss the limitations and advantages of this approach to discovery of interneurons that regulate motor behavior.     

The candidate tumor suppressor SASH1 interacts with the actin cytoskeleton and stimulates cell-matrix adhesion

SASH1, a member of the SLY-family of signal adapter proteins, is a candidate tumor suppressor in breast and colon cancer. Reduced expression of SASH1 is correlated with aggressive tumor growth, metastasis formation, and inferior prognosis. However, the biological role of SASH1 remains largely unknown. To unravel the function of SASH1, we have analyzed the intracellular localization of endogenous SASH1, and have generated structural SASH1 mutants. SASH1 localized to the nucleus as well as to the cytoplasm in epithelial cells. In addition, SASH1 was enriched in lamellipodia and membrane ruffles, where it co-distributed with the actin cytoskeleton. Moreover, we demonstrate a novel interaction of SASH1 with the oncoprotein cortactin, a known regulator of actin polymerization in lamellipodia. Enhanced SASH1 expression significantly increased the content of filamentous actin, leading to the formation of cell protrusions and elongated cell shape. This activity was mapped to the central, evolutionarily conserved domain of SASH1. Furthermore, expression of SASH1 inhibited cell migration and lead to increased cell adhesion to fibronectin and laminin, whereas knock-down of endogenous SASH1 resulted in significantly reduced cell–matrix adhesion. Taken together, our findings unravel for the first time a mechanistic role for SASH1 in tumor formation by regulating the adhesive and migratory behaviour of cancer cells.     

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.     

F<Subscript>1</Subscript>F<Subscript>0</Subscript>-ATP Synthase Complex Interactions <Emphasis Type="Italic">In Vivo</Emphasis> Can Occur in the Absence of the Dimer Specific Subunit e

Evidence suggests membrane bound F₁F₀-ATPase complexes form stable associations such that dimers can be retrieved from detergent lysates of mitochondria isolated from a range of sources including algae, higher plants, yeast and bovine heart, and plant chloroplasts. The physiological relevance of these interactions is not clear but may be connected with the formation and structure of mitochondrial cristae. We sought to demonstrate, in vivo, the association of F₁F₀-ATPases in yeast cells co-expressing two b subunits each fused at its C-terminus to a GFP variant appropriate for fluorescence resonance energy transfer (FRET; BFP as the donor and GFP as the acceptor fluorophore). Both subunit b-GFP and b-BFP fusions were assembled into functional complexes. FRET was observed from enzyme complexes in molecular proximity in respiring cells providing the first demonstration of the association, in vivo, of F₁F₀-ATPase complexes. Moreover, FRET was observed within cells lacking the dimer specific subunit e, indicating structured associations can occur within the inner membrane in the absence of subunit e.     

GFP-reporter for a high throughput assay to monitor estrogenic compounds

In vitro test systems using yeast cells are a useful tool for the determination of the estrogenic activity of estrogens, phyto- and xeno-estrogens and can be used for monitoring large sample numbers in a routine analysis procedure. Our conventional transactivation assay functions with an expression plasmid expressing estrogen receptor α (ERα) under the control of a copper-inducible CUP1 promoter and a reporter plasmid expressing β-galactosidase under the control of the vitellogenin estrogen response element (ERE). In the novel yeast screen system the lacZ gene in the reporter plasmid was substituted by a gene for green fluorescent protein (GFP). Incubation of yeast with various concentrations of estrogenically active substances led to expression of the reporter gene product GFP in a dose dependent manner. The yeast transactivation assay was further down-scaled to be performed in a microplate scale, which is an important step to facilitate handling of large sample numbers. The sensitivity and reproducibility of the novel test system could be confirmed by analysis of the potencies of various estrogenically active substances. Thus, the newly developed yeast estrogen screen using GFP as a reporter can substitute the assay that has been used for a period of several years.     

Selective recruitment of <Emphasis Type="Italic">Adh</Emphasis> genes for distinct enzymatic functions in <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Alcohol dehydrogenase (ADH) activity in plants is generally associated with glycolytic fermentation, which facilitates cell survival during episodes of low-oxygen stress in water-logged roots as well as chronically hypoxic regions surrounding the vascular core. Work with tobacco and potato has implicated ADH activity in additional metabolic roles, including aerobic fermentation, acetaldehyde detoxification and carbon reutilization. Here a combination of approaches has been used to examine tissue-specific patterns of Adh gene expression in order to provide insight into the potential roles of alcohol dehydrogenases, using Petunia hybrida, a solanaceous species with well-characterized genetics. A reporter-gene study, relying on the promoters of Adh1 and Adh2 to drive expression of the gene for a green fluorescent protein derivative, mgfp5, revealed unexpectedly complex patterns of GFP fluorescence in floral tissues, particularly the stigma, style and nectary. Results of GC-MS analysis suggest the association of ADH with production of aromatic compounds in the nectary. Overall the results demonstrate selective recruitment of Adh gene family members in tissues and organs associated with diverse ADH functions.     

The evolution of host use and unusual reproductive strategies in Achrysocharoides parasitoid wasps

We studied host selection and exploitation, two crucial aspects of parasite ecology, in Achrysocharoides parasitoid wasps, which show remarkable host specificity and unusual offspring sex allocation. We estimated a molecular phylogeny of 15 Achrysocharoides species and compared this with host (plant and insect) phylogenies. This tri-trophic phylogenetic comparison provides no evidence for cospeciation, but parasitoids do show phylogenetic conservation of the use of plant genera. Patterns of sequence divergence also suggest that the parasitoids radiated more recently (or evolved much faster) than their insect hosts. Three main categories of brood production occur in parasitoids: (1) solitary offspring, (2) mixed sex broods and (3) separate (split) sex broods. Split sex broods are very rare and virtually restricted to Achrysocharoides, while the other types occur very widely. Our phylogeny suggests that split sex broods have evolved twice and provides evidence for a transition from solitary to mixed sex broods, via split sex broods, as predicted by theory.