PKA在盘基网柄菌(Dictyostelium discoideum)多细胞发育中的作用

在盘基网柄菌(Dictyosteliumdiscoideum)多细胞发育中,蛋白激酶A(proteinkinaseA,PKA)发挥多重作用.细胞聚集阶段,PKA调节腺苷酰环化酶的活性,中转cAMP,诱导dut、pdi等一些发育早期的基因表达;参与启动聚集后的细胞分化和形态构成,增强GBF活性,激活前孢子细胞特有基因的表达;它还精密调控前柄细胞特有基因ecmB的表达,准确启动拔顶发育,诱导孢柄和孢子的成熟.子实体形成后,PKA又是维持孢子休眠和保证孢子有效萌发的必需因子.在PKA调控下,盘基网柄菌有条不紊地完成整个发育过程.     

将盘基网柄菌用于本科细胞生物学综合性实验教学初探——电穿孔转化绿色荧光蛋白

盘基网柄菌(Dictyostelium discoideum)是一个应用广泛的模式生物,非常适合用来研究胞质分裂、细胞运动、吞噬作用、趋化性、趋电性、信号转导以及个体发育过程中的细胞分化。该实验主要介绍电穿孔技术转化绿色荧光蛋白标记肌动蛋白基因质粒(Lifeact-GFP)进入盘基网柄菌活细胞中,抗性筛选(潮霉素B)获得阳性克隆子,最后借助荧光显微镜观察绿色荧光蛋白标记的微丝在盘基网柄菌的分布情况。综合性实验训练可提高学生的学习兴趣和综合运用理论知识的能力,进而可培养学生的科学研究思维。     

盘基网柄菌(Dictyostelium discoideum)细胞的分化及其调控

本文综述了盘基网柄菌(Dictyostelium dis-coideum)发育过程中细胞类型的诱导和分化,细胞外cAMP及其四种位于细胞表面的受体及PKA(蛋白激酶A)、GSK-3(糖原合成酶激酶)和STATa等在网柄菌发育过程中的作用。     

盘基网柄菌中分化诱导因子信号与细胞命运决定

对盘基网柄菌发育过程中分化诱导因子(DIF)的作用及其机制进行了综述,包括DIF对盘基网柄菌前柄细胞、柄细胞分化的作用以及DIF的生物合成、DIF的诱导、降解失活、DIF对细胞命运和细胞比例的调节及其作用机制等。     

盘基网柄菌发育中的细胞粘附分子及其信号转导

在盘基网柄菌发育早期,DdCAD-1和csA调节了变形虫细胞间的粘着,调控该过程的机制类似于胚胎发育中上皮细胞层的闭合。完成网柄菌发育的一个必需分子是gpl50异嗜性粘附分子。盘基网柄菌β-连环蛋白同源物Aardvark(Aar)的缺乏使细胞间失去粘着连接,Aar也有信号转导功能,调控了前孢子细胞基因的表达。因此,细胞间的粘着是盘基网柄菌发育的一个重要组成部分,并与调控形态发生过程的信号转导有密切相互作用关系。     

细胞色素c在盘基网柄菌细胞凋亡中的作用

细胞色素c在细胞凋亡中发挥着重要的作用,其作用机理在高等真核生物及低等真核生物酵母中已经比较清楚,但在盘基网柄菌(Dictyostelium discoideum)中的作用却没有相关报道.所以我们用western blot和实时荧光定量PCR的方法分别测定了盘基网柄菌前柄细胞和前孢子细胞中细胞色素c的含量及表达量的变化...     

采自广西弄岗自然保护区的柄杯菌属真菌

依据广西弄岗自然保护区采集的标本,鉴定出柄杯菌属真菌4种,即麦里西柄杯菌(Podoscypha mellissii)、内卷柄杯菌(P. involuta)、巴西柄杯菌(P. brasiliensis)和长喙柄杯菌(P. gillesii)。其中,长喙柄杯菌为中国新记录种,巴西柄杯菌首次在中国大陆发现,文中提供了该2种详细形态特征描述及图片信息。     

集胞菌的初步研究

本文报告了网柄菌属(Dictyostelium)的4个种:毛霉状网柄菌(D.mucoroides Brefeld)、小网柄菌(D.minutum Raper)、紫网柄菌(D.purpureum Olive)和盘基网柄菌(D.discoideumRaper)。轮柄菌属(Polysphondylium)的1个种,即紫轮柄菌(P.violaceum Brefeld)。共5个种,以上均为我国新记录。此外,又增补了一些分离培养的方法,对每个种的生活史进行了观察,并提出了小囊胞应纳入生活史中。     

gp150蛋白在盘基网柄菌发育中的作用及粘附分子间关系的分析

饥饿的盘基网柄菌进入多细胞发育期 ,在发育早期 ,AK12 7细胞 (gp150突变细胞 )能表达DdCAD 1和 gp80两种粘附分子 ,但它们不足以促进细胞继续发育 ,发育停留在细胞疏松结合阶段。粘附分子 gp150调节的细胞与细胞间的粘着影响了细胞丘“突出”的形成 ,由此影响了盘基网柄菌多细胞发育的形态发生。TL93细胞 (DdCAD 1和gp80突变细胞 )能完成发育。主要原因是在细胞流发育阶段就表达了gp150分子 ,在细胞粘着的功能上有替代DdCAD 1和 gp80的作用。因此 gp150蛋白对盘基网柄菌多细胞发育有着不可或缺的作用     

中国韧革菌(Ⅲ)

本文报道中国韧革菌科(Stereaceae Pilat)真菌17种,隶属于柄革菌属(Stereopsis Reid)。韧革菌属(Stereum Hill:Persoon)和刷革菌属(Xylobolus Karsten)。其中有3个新种,1个新组合种和2个新记录种,它们是:厚盖柄革菌(Stereopsis crassipileata Z.T.Guo,sp.nov.)、细柄柄革菌(S.gracilistipitata Z.T.Guo,sp.nov.),假杯柄革菌(S.pseudocupulata Z.T.Guo,sp。nov.)、掌状柄革菌(S.craspedia(Fr.)Z.T.Guo,comb.nov.)、瓣裂柄革菌(S.hiscens(Berk. & Rav.)Reid)和蛋黄柄革菌(S.uitellina(Plowr.)Reid)。标本全部保藏于中国科学院微生物研究所真菌标本室(HMAS)。     

Dual-laser, differential fluorescence correction method for reducing cellular background autofluorescence

A method has been developed for reducing the intrinsic autofluorescence background component in cells labeled with fluorescent antibodies, thus permitting low levels of antibody-binding on highly autofluorescent cells to be quantified. The method is based on the broad autofluorescent excitation spectra compared to the well-defined spectra of the fluorescent label. Two laser wavelengths were used, one optimally to excite the fluorescent label plus autofluorescence and the second to excite only the autofluorescence. Two fluorescence measurements were made in the same wavelength region and the signals were subtracted on a cell-by-cell basis using a difference amplifier to zero the autofluorescence and amplify the signal from the fluorescent label. Test results on unlabeled autofluorescent macrophages showed that the autofluorescence component was reduced by balancing the signal inputs to the difference amplifier. When labeled macrophages were analyzed, the autofluorescence was reduced and the fluorescent-labeled antibody-binding component was amplified. The method was also able to resolve labeled lymphocytes from unlabeled autofluorescent macrophages.     

A single laser method for subtraction of cell autofluorescence in flow cytometry

In flow cytometry cell autofluorescence often interferes with efforts to measure low levels of bound fluorescent antibody. We have developed a way to correct for autofluorescence on a cell-by-cell basis. This results in improved estimates of real staining and better separation of the fluorescence histograms of stained and non-stained cells. Using a single laser, two-color fluorescence measurement system and two-color compensation electronics, autofluorescence and one fluorescent reagent are measured (rather than two fluorescent reagents). With fluorescein-conjugated antibodies the signal in the 515 to 555 nm range (green fluorescence) includes both fluorescein emission and part of the cellular autofluorescence. In the cases we have investigated, autofluorescence collected at wavelengths above 580 nm ("red") is well correlated with the green autofluorescence of the cells. A fraction of this red fluorescence is subtracted from the green fluorescence to produce an adjusted fluorescein output on which unstained cells have zero average signal. Use of this method facilitates the selection of rare cells transfected with surface antigen genes. Culture conditions affect the level of autofluorescence and the balance between red and green autofluorescence. When applied with fluorescein-conjugated reagents, the technique is compatible with the use of propidium iodide for live/dead cell discrimination.     

Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella

The soil amoeba Dictyostelium discoideum is a haploid eukaryote that, upon starvation, aggregates and enters a developmental cycle to produce fruiting bodies. In this study, we infected single-cell stages of D. discoideum with different Legionella species. Intracellular growth of Legionella in this new host system was compared with their growth in the natural host Acanthamoeba castellanii . Transmission electron microscopy of infected D. discoideum cells revealed that legionellae reside within the phagosome. Using confocal microscopy, it was observed that replicating, intracellular, green fluorescent protein (GFP)-tagged legionellae rarely co-localized with fluorescent antibodies directed against the lysosomal protein DdLIMP of D. discoideum . This indicates that the bacteria inhibit the fusion of phagosomes and lysosomes in this particular host system. In addition, Legionella infection of D. discoideum inhibited the differentiation of the host into the multicellular fruiting stage. Co-culture studies with profilin-minus D. discoideum mutants and Legionella resulted in higher rates of infection when compared with infections of wild-type amoebae. Because the amoebae are amenable to genetic manipulation as a result of their haploid genome and because a number of cellular markers are available, we show for the first time that D. discoideum is a valuable model system for studying intracellular pathogenesis of microbial pathogens.     

Development of a quantitative diagnostic method of estrogen receptor expression levels by immunohistochemistry using organic fluorescent material-assembled nanoparticles

The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3′-diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature and substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues sections to quantitatively examine the two methods. The results demonstrated that our nanoparticle staining analyzed a wide range of ER expression levels with higher accuracy and quantitative sensitivity than DAB staining. This enhancement in the diagnostic accuracy and sensitivity for ERs using our immunostaining method will improve the prediction of responses to therapies that target ERs and progesterone receptors that are induced by a downstream ER signal.     

Evaluation of different methods detecting intracellular generation of free radicals

Reactive oxygen species (ROS) play several biological roles. We investigated the applicability of fluorescent probes for their detection (i) in rabbit lens epithelial cells during ageing in culture, and (ii) in thin sections of rat heart. We used dihydroethidium (DHE), dichlorofluorescin (DCFH), and dihydrorhodamine 123 (DHR) together with detection of autofluorescence both in cells and in chloroform extracts. Superoxide production was confirmed by a specific histochemical method using Mn²⁺. All methods demonstrated higher production of ROS in older cells. All probes revealed different sites of ROS production in young and old cells and could be used for investigation of ROS generation during cell ageing. In the thin sections of rat heart DCFH was not suitable for intracellular ROS detection. The results indicate that the potential of fluorescent dyes in ROS detection is not usually fully exploited, and that blue autofluorescence is associated with oxidative damage.     

Improved in vivo whole-animal detection limits of green fluorescent protein-expressing tumor lines by spectral fluorescence imaging

Green fluorescent protein (GFP) has been used for cell tracking and imaging gene expression in superficial or surgically exposed structures. However, in vivo murine imaging is often limited by several factors, including scatter and attenuation with depth and overlapping autofluorescence. The autofluorescence signals have spectral profiles that are markedly different from the GFP emission spectral profile. The use of spectral imaging allows separation and quantitation of these contributions to the total fluorescence signal seen in vivo by weighting known pure component profiles. Separation of relative GFP and autofluorescence signals is not readily possible using epifluorescent continuous-wave single excitation and emission bandpass imaging (EFI). To evaluate detection thresholds using these two methods, nude mice were subcutaneously injected with a series of GFP-expressing cells. For EFI, optimized excitation and emission bandpass filters were used. Owing to the ability to separate autofluorescence contributions from the emission signal using spectral imaging compared with the mixed contributions of GFP and autofluorescence in the emission signal recorded by the EFI system, we achieved a 300-fold improvement in the cellular detection limit. The detection limit was 3 x 10(3) cells for spectral imaging versus 1 x 10(6) cells for EFI. Despite contributions to image stacks from autofluorescence, a 100-fold dynamic range of cell number in the same image was readily visualized. Finally, spectral imaging was able to separate signal interference of red fluorescent protein from GFP images and vice versa. These findings demonstrate the utility of the approach in detecting low levels of multiple fluorescent markers for whole-animal in vivo applications.     

Quantitative comparison of the sensitivity of detection of fluorescent and bioluminescent reporters in animal models

Bioluminescent and fluorescent reporters are finding increased use in optical molecular imaging in small animals. In the work presented here, issues related to the sensitivity of in vivo detection are examined for standard reporters. A high-sensitivity imaging system that can detect steady-state emission from both bioluminescent and fluorescent reporters is described. The instrument is absolutely calibrated so that animal images can be analyzed in physical units of radiance allowing more quantitative comparisons to be performed. Background emission from mouse tissue, called autoluminescence and autofluorescence, is measured and found to be an important limitation to detection sensitivity of reporters. Measurements of dual-labeled (bioluminescent/fluorescent) reporter systems, including PC-3M-luc/DsRed2-1 and HeLa-luc/PKH26, are shown. The results indicate that although fluorescent signals are generally brighter than bioluminescent signals, the very low autoluminescent levels usually results in superior signal to background ratios for bioluminescent imaging, particularly compared with fluorescent imaging in the green to red part of the spectrum. Fluorescence detection sensitivity improves in the far-red to near-infrared, provided the animals are fed a low-chlorophyll diet to reduce autofluorescence in the intestinal region. The use of blue-shifted excitation filters is explored as a method to subtract out tissue autofluorescence and improve the sensitivity of fluorescent imaging.     

Microspectroscopic imaging of nodulation factor-binding sites on living Vicia sativa roots using a novel bioactive fluorescent nodulation factor.

A novel bioactive fluorescent nodulation (Nod) factor, NodRlv-IV(BODIPY FL-C16), has been synthesized by attaching a BODIPY FL-C16 acyl chain to the primary amino group of chitotetraose deacetylated at the nonreducing terminus by recombinant NodB. The binding of the fluorescent Nod factor to root systems of Vicia sativa was investigated with fluorescence spectral imaging microscopy (FSPIM) and fluorescence ratio imaging microscopy (FRIM). Spatially resolved fluorescence spectra of living and labeled Vicia sativa root systems were measured by FSPIM. Strong autofluorescence, inherent to many plant systems when excited at 488 nm, was corrected for by utilizing the difference in fluorescence emission spectra of the autofluorescence and NodRlv-IV(BODIPY FL-C16). A methodology is presented to break down the in situ fluorescence emission spectra into spatially resolved autofluorescence and BODIPY FL fluorescence spectra. Furthermore, an FRIM method was developed for correcting autofluorescence in fluorescence micrographs for this system. After autofluorescence correction it was shown that NodRlv-IV(BODIPY FL-C16) was concentrated in the root hairs, but was also bound to other parts of the root surface.     

Function of the Dictyostelium discoideum Atg1 kinase during autophagy and development

When starved, the amoebae of Dictyostelium discoideum initiate a developmental process that results in the formation of fruiting bodies in which stalks support balls of spores. The nutrients and energy necessary for development are provided by autophagy. Atg1 is a protein kinase that regulates the induction of autophagy in the budding yeast Saccharomyces cerevisiae. In addition to a conserved kinase domain, Dictyostelium Atg1 has a C-terminal region that has significant homology to the Caenorhabditis elegans and mammalian Atg1 homologues but not to the budding yeast Atg1. We investigated the function of the kinase and conserved C-terminal domains of D. discoideum Atg1 (DdAtg1) and showed that these domains are essential for autophagy and development. Kinase-negative DdAtg1 acts in a dominant-negative fashion, resulting in a mutant phenotype when expressed in the wild-type cells. Green fluorescent protein-tagged kinase-negative DdAtg1 colocalizes with red fluorescent protein (RFP)-tagged DdAtg8, a marker of preautophagosomal structures and autophagosomes. The conserved C-terminal region is essential for localization of kinase-negative DdAtg1 to autophagosomes labeled with RFP-tagged Dictyostelium Atg8. The dominant-negative effect of the kinase-defective mutant also depends on the C-terminal domain. In cells expressing dominant-negative DdAtg1, autophagosomes are formed and accumulate but seem not to be functional. By using a temperature-sensitive DdAtg1, we showed that DdAtg1 is required throughout development; development halts when the cells are shifted to the restrictive temperature, but resumes when cells are returned to the permissive temperature.     

Quenching autofluorescence in the alimentary canal tissues of Bactericera cockerelli (Hemiptera: Triozidae) for immunofluorescence labeling

Immunofluorescence has been widely used to localize microbes or specific molecules in insect tissues or cells. However, significant autofluorescence is frequently observed in tissues which can interfere with the fluorescent identification of target antigens, leading to inaccurate or even false positive fluorescent labeling. The alimentary canal of the potato psyllid, Bactericera cockerelli ?ulc, exhibits intense autofluorescence, hindering the application of immunolocalization for the detection and localization of the economically important pathogen transmitted by this insect, “Candidatus Liberibacter solanacearum” (Lso). In the present study, we tested the use of irradiation, hydrogen peroxide (H₂O₂) and Sudan black B (SBB) treatments to reduce the autofluorescence in the B. cockerelli alimentary canal tissues. Furthermore, we assessed the compatibility of the above‐mentioned treatments with Lso immunolocalization and actin staining using phalloidin. Our results showed that the autofluorescence in the alimentary canal was reduced by irradiation, H₂O₂, or SBB treatments. The compatibility assays indicated that irradiation and H₂O₂ treatment both greatly reduced the fluorescent signal associated with Lso and actin. However, the SBB incubation preserved those target signals, while efficiently eliminating autofluorescence in the psyllid alimentary canal. Therefore, herein we propose a robust method for reducing the autofluorescence in the B. cockerelli alimentary canal with SBB treatment, which may improve the use of immunofluorescence labeling in this organism. This method may also have a wide range of uses by reducing the autofluorescence in other arthropod species.