Bidirectional membrane molecule transfer between dendritic and T cells

The acquisition of dendritic cell (DC) molecules by T cells has been previously reported. However, it remains unclear whether the transfer is only mono- or bidirectional. In this study, we incubated CMFDA-labeled ovalbumin (OVA)-pulsed DC2.4 (DC2.4(OVA)) cells with Dil-labeled OT II CD4(+) T cells and analyzed the potential bidirectional molecule transfer. We also assessed the distribution of internalized membrane using two engineered DC2.4/Ia(b)GFP and MF4/TCRCFP DC lines. Our findings showed that membrane molecule transfer is bidirectional. CD4(+) T cells acquired Ia(b), CD11c, CD40, and CD80 from DC2.4(OVA) cells, and conversely DC2.4(OVA) cells took up CD4, CD25, CD69, and T cell receptor from T cells. The internalized molecules acquired by T cells and DCs mostly localized in endosomes and lysosomes, respectively. Taken together, this study demonstrated a novel phenomenon of bidirectional membrane molecule transfer between DCs and T cells.     

A flow-cytometric method for determination of yeast viability and cell number in a brewery

A flow-cytometric assay, using the fluorescent dye, oxonol, for the simultaneous determination of yeast cell viability and cell number is described. The assay was optimised, and trialed at a brewery for 6 months. The flow-cytometry assay offered a substantially reduced error in viability determination, compared to methylene blue which is the industry standard for measuring viability. Further, by calculating yeast cell number at the same time, this assay provides a reliable method for determining pitching rate, allowing increased quality control of subsequent fermentations.     

Membrane topography of HLA I, HLA II, and ICAM-1 is affected by IFN-gamma in lipid rafts of uveal melanomas

The lateral distribution and colocalization of HLA I, HLA-DR, and ICAM-1 proteins was studied for the first time in the plasma membrane of two human uveal melanoma cell lines, OCM-1 and OCM-3. Our fluorescence resonance energy transfer and confocal laser scanning microscopic experiments revealed that these molecules are mostly confined to the same membrane regions, where they form similar protein patterns (homo- and hetero-associates) to those found previously on other cell types of lymphoid as well as colorectal carcinoma origin. Confocal microscopic colocalization experiments with GM(1) gangliosides and the GPI-anchored CD59 molecules showed enrichment of HLA I, HLA-DR, and ICAM-1 molecules in specific membrane domains (lipid rafts) excluding the transferrin receptor. IFN-gamma remarkably increased the expression levels of these molecules and rearranged their association patterns, which can affect the adoptive immune response of effector cells.     

Quantification of cell hybridoma yields with confocal microscopy and flow cytometry

The fusion of antigen presenting and cancer cells leads to the formation of hybrid cells, which are considered a potential vaccine for treating cancer. The quality assessment of hybrid cell vaccines is crucial for the introduction of this new treatment. Flow cytometry was the method used recently, since it is faster in comparison to classical microscopy. Here we describe a rapid confocal microscopy based approach to quantify hybrid cell yields. The extent of fusion rate was determined by confocal microscopy by counting dual fluorescent cells and by measuring the area of co-localized pixels. Results of both methods showed high degree of correlation. The same samples were also analyzed by flow cytometry. Fusion rates determined with both techniques showed significant correlation. In conclusion, using confocal microscopy we developed a sensitive and a rapid method to assess the yield of hybridomas in a large number of electrofused cells.     

Confocal microscopic observation of fusion between baculovirus budded virus envelopes and single giant unilamellar vesicles

We assayed fusion events between giant unilamellar vesicles (GUVs) and budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus), the envelopes of which have been labeled with the fluorescent dye Alexa Fluor 488. This involves observing the intensity of fluorescence emitted from the lipid bilayer of single GUVs after fusion using laser scanning microscopy. Using this assay system, we found that fusion between single GUVs and BV envelopes was significantly enhanced at around pH 5.0-6.0, which suggests that: (1) envelope glycoprotein GP64-mediated membrane fusion within the endosome of insect cells was reproduced in our artificial system; (2) acidic phospholipids in GUVs are necessary for this fusion, which are in agreement with the previous results with conventional small liposomes including large unilamellar vesicles and multilamellar vesicles; and (3) the efficiency of fusion is significantly affected by membrane properties that can be modulated by adding cholesterol to GUV lipid bilayers. In addition, the microscopic observation of BV-fused single GUVs showed that a weak interaction occurred between BVs and GUVs containing dioleoylphosphatidylserine at pH 6.0-6.5, and components of BV envelopes were unevenly distributed upon fusion with GUVs containing saturated phospholipid with cholesterol. We further demonstrated that when the recombinant membrane protein, adrenergic β₂ receptor, was expressed on recombinant BV envelopes, the protein distribution on BV-fused GUVs was also affected by their lipid contents.     

Multiparametric flow cytometry allows rapid assessment and comparison of lactic acid bacteria viability after freezing and during frozen storage

Freezing is widely used for the long-term preservation of lactic acid bacteria, but often affects their viability and technological properties. Different methods are currently employed to determine bacterial cryotolerance, but they all require several hours or days before achieving results. The aim of this study was to establish the advantages of multiparametric flow cytometry by using two specific fluorescent probes to provide rapid assessment of the viability of four strains of Lactobacillus delbrueckii after freezing and during frozen storage. The relevance of carboxyfluorescein diacetate and propidium iodide to quantify bacterial viability was proven. When bacterial suspensions were simultaneously stained with these two fluorescent probes, three major subpopulations were identified: viable, dead and injured cells. The cryotolerance of four L. delbrueckii strains was evaluated by quantifying the relative percentages of each subpopulation before and after freezing, and throughout one month of storage at -80 degrees C. Results displayed significant differences in the resistance to freezing and frozen storage of the four strains when they were submitted to the same freezing and storage procedures. Whereas resistant strains displayed less than 10% of dead cells after one month of storage, one sensitive strain exhibited more than 50% of dead cells, together with 14% of stressed cells after freezing. Finally, this study proved that multiparametric flow cytometry was a convenient and rapid tool to evaluate the viability of lactic acid bacteria, and was well correlated with plate count results. Moreover, it made it possible to differentiate strains according to their susceptibility to freezing and frozen storage.     

四唑盐减低法结合激光共聚焦显微镜定量分析表皮葡萄球菌生物被膜体外模型

目的建立体外表皮葡萄球菌(Staphylococcus epidermidis)生物膜(biofilm,BF)模型,观察和定量分析表皮葡萄球菌BF的动态形成过程。方法采用可形成BF的表皮葡萄球菌RP62A,平板法建立体外BF模型,四唑盐(tetrazolium salt,XTT)减低法定量检测BF形成过程中细菌活力的变化,激光共聚焦显微镜(confocal laser scan-ning microscopy,CLSM)结合图像结构分析软件(image structure analyer,ISA)对BF形成过程结构参数进行动态分析,扫描电镜(scanning electron microscope,SEM)观察BF形成过程中的形态结构。结果在12、24和48 h时,XTT减低法A450的值分别为2.39±0.48、3.41±0.18和3.92±0.27,P0.05;ISA软件定量分析显示在区域孔径(AP)的值分别为0.84±0.08、0.68±0.01和0.59±0.13,P0.05,平均扩散距离(ADD)的值分别为1.34±0.24、1.49±0.09和1.89±0.39,P0.05,结构熵(TE)的值分别为4.71±0.82、8.69±0.68和8.94±0.28,24 h、48 h与12 h相比,P0.05。结论表皮葡萄球菌BF的形成是个动态的过程,24 h时BF基本形成,48 h BF结构更加复杂。XTT减低法,CLSM结合ISA软件,SEM三种方法联合使用是观察和定量分析体外BF模型较理想的方法。     

The somatic musculature of Bryceella stylata (Milne, 1886) (Rotifera: Proalidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology

The monogonont rotifer Bryceella stylata was investigated with light, electron and confocal laser scanning (CLSM) microscopy to provide detailed insights into its anatomy and new information for future phylogenetic analyses of the group. Results from CLSM and phalloidin staining revealed a total of six paired longitudinal muscles (musculi longitudinales I-VI) and eight circular muscles (musculi circulares I-VIII) as well a complex network of mostly fine visceral muscles. In comparison with other rotifer species that have been investigated so far, B. stylata shares the presence of the circular and longitudinal muscles: musculus longitudinalis ventralis, musculus longitudinalis lateralis inferior, musculus longitudinalis dorsalis, musculus longitudinalis capitis and musculus circumpedalis. However, the species lacks lateral and dorsolateral longitudinal muscles and some circular muscles (e.g., corona sphincter, musculus pars coronalis). With light and electron microscopy, we were able to document the precise number of pseudosegments and the arrangement of the chambers comprising the trophi elements. Furthermore, our observations revealed several new morphological characteristics, including a shield-like epidermal projection covering the dorsal antenna, an epidermal projection restricting the corona caudally and an unpaired hypopharynx with distinct shovel-like structures.     

Impairment of anterograde and retrograde neurofilament transport after anti-kinesin and anti-dynein antibody microinjection in chicken dorsal root ganglia

The purpose of the present study was to investigate the participation of the motor proteins kinesin and dynein in axonal transport of neurofilaments (NF) in cultured dorsal root ganglia neurons. Therefore, we performed live-recording studies of the green fluorescent protein-tagged neurofilament M (GFP-NF-M) to assay transport processes in neurons. Co-localization studies revealed that GFP-NF-M was capable to build a functional NF network with other NF subunits, including phosphorylated heavy neurofilaments (NF-H-PH). Time-lapse recordings using confocal laser scanning microscopy exhibited fast transport of NF dots in anterograde and retrograde direction through a photobleached gap. Following microinjection of anti-kinesin antibodies or colchicine treatment an impairment of anterograde as well as retrograde NF transport was observed during live-recording experiments. In contrast, microinjection of anti-dynein antibodies only impaired retrograde transport of NF whereas the anterograde movement of GFP-NF-M was unaffected. Treatment of the cells with unspecific antibodies had no effect.     

Mast cell degranulating peptide binds to RBL-2H3 mast cell receptors and inhibits IgE binding

Fluorescent and biotinylated analogs of mast cell degranulating (MCD) peptide were synthesized and the labels fluoresceinisothiocyanate and N-hydroxysuccinimidobiotin were conjugated at position 1 in the MCD peptide sequence. The analogs with these moieties retained histamine-releasing activity as high as that of the parent MCD peptide in rat peritoneal mast cell assays. These labeled analogs were used in rat basophilic leukemia cells (RBL-2H3) to demonstrate by confocal microscopy and flow cytometry the specific binding of MCD peptide to mast cell receptors. Consequently MCD peptide was found to compete with and inhibit the binding of fluorescent IgE on RBL cells as monitored by flow cytometry. Thus MCD peptide may prove to be useful in the study of IgE receptor-bearing cells.     

Functional cloning of the dihydropteroate synthase gene of Staphylococcus haemolyticus

Abstract The usefulness of oxonol ( bis -(1,3-dibutylbarbituric acid)trimethine oxonol) as a generally applicable indicator of bacterial viability was investigated using untreated and killed cultures of a variety of bacterial genera. Killing methods involved either heat or bactericidal antibiotics. For all strains tested, the fluorescent dye showed significantly more intense staining of killed than untreated cells. The sensitivity of Aeromonas salmonicida to gentamicin was assessed using oxonol. Although the bacterium was shown to be sensitive to the antibiotic, there was a delay between the time cells lost culturability, as judged by numbers of colony forming units, and that for which a dead cell population could be detected by flow cytometry.     

Megagametophyte abnormalities of near-isogenic female partial-sterile soybean mutants (Glycine max; Leguminosae)

Megagametogenesis of soybean, Glycine max (L.) Merr., cultivars Clark and Clark k2, and F1 hybrid of Clark (female parent) crossed with Clark k2 (male parent) were studied using stereo light microscopy and confocal scanning laser microscopy. Reproductive development in Clark and Clark k2 plants was compared to F1 hybrid plants. In mature pods, 6.4% of the ovules of Clark, 8.1% of the ovules of Clark k2, and 41.4% of the ovules of F1 hybrid plants were aborted. This female partial sterility was due to incomplete megagametophyte development: undeveloped polar nuclei—or developed but not in a position for fertilization; increased megagametophyte wall thickness; abnormal shape and/or premature degeneration of synergids and intact synergids throughout the life of the ovule; egg cell not well-developed or absent; and megagametophyte remaining uninucleate. Each of these abnormalities contributed to either lack of double fertilization or early megagametophyte abortion. Electronic Publication     

Specific lectins map the distribution of fibronectin and beta 1-integrin on living MDCK cells

The expression and dynamics of bound fibronectin and the sialylated integral membrane protein, beta 1-integrin, were analyzed on the apical membrane of living MDCK cells. Fibronectin was identified by its specific binding of fluorescent peanut agglutinin and sialylated beta 1-integrin by its binding of Sambucus nigra agglutinin. Confocal epifluorescence microscopy and laser scanning cytometry determined the distribution and abundance of binding sites of the two fluorescently labeled lectins. Both fibronectin and beta 1-integrin were restricted to specific regions uniformly distributed over the entire apical surface. Apical-surface fibronectin binding varied much more between cells than did the expression of beta 1-integrin. Sialylated beta 1-integrin colocalized >92% with membrane microplicae while fibronectin was unrelated to these surface structures. This lack of colocalization of the proteins was confirmed by double-labeling experiments. From the maturation dependence of the fibronectin-binding capacity and the differences in protein turnover times, it was evident that fibronectin did not bind to sialylated beta 1-integrin. Furthermore, desialylation of beta 1-integrin uncovered additional fibronectin receptors on the apical membrane. We conclude that these lectins permit tracking of two membrane-associated glycoproteins in living cells and that fibronectin binds only to desialylated beta 1-integrin on MDCK cells.     

原代培养脊髓神经元线状溶酶体与细胞骨架蛋白-纽蛋白关系的研究

目的研究原代培养脊髓神经元线状溶酶体(nematolysosome)的形成与分布及其与细胞骨架蛋白-纽蛋白(vinculin)的关系.方法用细胞松弛素D(cytochalasin D,CD)及佛波醇酯(phorbol myristate acetate,PMA)处理原代培养脊髓神经元,用免疫荧光双标记纽蛋白及组织蛋白酶D(cathepsin D)、酸性磷酸酶(ACPase)、电镜细胞化学及共焦激光扫描显微镜方法研究线状溶酶体与纽蛋白的关系.结果在正常对照组神经元,组织蛋白酶D(标记溶酶体)与纽蛋白分布于胞质及突起内;在CD及PMA处理神经元,纽蛋白及组织蛋白酶D的分布呈向心性移动,但集聚的部位不同;电镜酶细胞化学方法显示CD组及PMA组神经元内线状溶酶体均增多.结论组织蛋白酶D及纽蛋白在培养脊髓神经元内协同分布,CD及PMA均可引起二者分布的变化,提示纽蛋白可通过增强细胞内吞体/溶酶体系统活动而使线状溶酶体增加,也可通过促进丝状肌动蛋白聚合而影响线状溶酶体的形成及运动.     

A study of the process of synchronisation and micronucleation in <Emphasis Type="Italic">Beta vulgaris</Emphasis> and the monitoring of an isolation procedure for micro-nuclei and micro-protoplasts by confocal laser scanning microscopy and flow cytometry

The process of synchronization and micro-nuclei induction in a suspension culture of Beta vulgaris, was induced by the sequential treatment with the DNA-synthesis inhibitor aphidicolin (30 μM, 24 h) and the spindle-toxin amiprophos-methyl (32 μM, 24 h). Mitotic arrest of divisions, spreading of G2-metaphase chromosomes, re-grouping of chromosomes, formation of a nuclear cell wall around single and re-grouped chromosomes and restitution of nuclei with a doubled DNA content was observed. The process of micro-nucleation was induced much less efficiently in Beta vulgaris than in Nicotiana plumbaginifolia. Cytological observations and monitoring of the process with flow cytometry and confocal laser scanning microscopy, was essential to follow up the course of events and to monitor the development of an efficient procedure for micro-protoplast isolation. Micro-nucleated protoplasts were fractioned by iso-osmotic Percoll gradient centrifugation to obtain heterogeneous micro-protoplast populations with cytoplasts and micro-protoplasts of different size. An enriched fraction with small sub-diploid micro-protoplasts was obtained with the equivalent DNA content of 1–4 chromosomes, as revealed by confocal laser scanning microscopy and flow cytometry. Sub-diploid micro-protoplasts with DNA amounts equivalent to 1–4 chromosomes were predominantly observed in the size classes of 1.8–10.2 μm at a frequency of 34.2–34.5%. The DNA measurements of micro-nuclei and micro-protoplasts, confirmed the hypothesis that the process of micro-nucleation followed the same course of cellular events as observed in N. plumbaginifolia. The correlation between DNA content and size of micro-nuclei and micro-protoplasts was not linear and affected by the degree of DNA condensation, total amount of DNA, and the presence of cytoplasm.     

Distribution of somatostatin receptor 5 in mouse and bullfrog retinas

Somatostatin (SRIF), as a neuroactive peptide in the CNS, may act as a neuromodulator through activation of five specific receptor subtypes (sst(1)-sst(5)). In this work we conducted a comparative study of the expression of sst(5) in mouse and bullfrog retinas by immunofluorescence double labeling. Basically, the expression profiles of sst(5) in the retinas of the two species were similar. That is, in the inner retina sst(5) was localized to dopaminergic and cholinergic amacrine cells, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) respectively, and cells in the ganglion cell layer, whereas in the outer retina immunostaining for sst(5) was observed in horizontal cells. However, a more widespread, abundant distribution of labeling for sst(5), as compared to mouse retina, was seen in bullfrog retina: strong labeling for sst(5) was diffusely distributed in both outer and inner plexiform layers (OPL and IPL) in the bullfrog retina, but the labeling was only observed in the IPL of the mouse retina. In addition, bullfrog photoreceptors, both rods and cones, but not mouse ones, were labeled by sst(5). In combination with the experiments showing that SRIF-immunoreactivity was mainly found in the inner retina, our results suggest that SRIF, released from SRIF-containing cells in the inner retina, may play a neuromodulatory role in both outer and inner retina mediated by volume transmission via sst(5) in bullfrog retina, while the SRIF action may be largely restricted to the mouse inner retina.