Fatty acid composition of four microsporidian species compared to that of their host fishes

ABSTRACT. The fatty acid composition of four microsporidian species (Glugea atherinae, Spraguea lophii, Glugea americanus , and Pleistophora mirandellae) and their host fishes has been determined using gas chromatography. Twenty-four fatty acids were identified with differences in relative abundance of fatty acids among the four parasites. Certain even-saturated fatty acids were found in a very high proportion: palmitic acid (16:0) represented one-third of total fatty acids in Pleistophora mirandellae. The level of docosahexaenoic acid (22:6ω3) attained 26–28% in Glugea atherinae, Spraguea lophii , and Glugea americanus , but only 8–9% in P. mirandellae. With respect to fatty acid compositions of host organs, some significant differences were evident between marine and freshwater fishes. Palmitic acid was prevalent in the marine fishes, Atherinae boyeri and Lophius piscatorius , and oleic acid (18:1ω9) in the freshwater fish Leuciscus cephalus. The proportion of docosahexaenoic acid in marine fishes was two or three times as great as in freshwater fish Leuciscus. The high polyunsaturated fatty acid content in both parasites and host fishes may be related to the scavenging of these fatty acids by the parasites rather than a microsporidia-specific fatty acid biosynthesis pathway.     

Development, Characterization and Future Prospects of Monoclonal Antibodies Against Spores of Glugea atherinae (Protozoa-Microsporidia-Fish Parasites)

ABSTRACT. Monoclonal antibodies against spores of Glugea atherinae were obtained after lymphocytic hybridization made from immunized mouse splenocytes. Screening using an indirect enzyme linked immunosorbent assay (ELISA), revealed seven monoclonal antibodies with an intense but variable reaction with the spores of fish microsporidia, and a moderate reaction with those of an insect microsporidium (Nosema sp.). The reaction was weaker with spores of Encephalitozoon intestinalis found in HIV' patients. FITC and Dot Blot confirmed the majority of these results. After biotinylation of the seven antibodies, inhibition tests allowed the localization of two different recognition domains on the spores of Glugea atherinae . The multiple antigenic determinants and their probable polysaccharide nature seem to be in accord with the class IgM of the antibodies produced. This work confirms the potential of these antibodies for microsporidian taxonomy and diagnosis, especially the use of Mabs 12F9 and 12H5 for detection of spores in stools of HIV⁺ patients.     

Flow Cytometric Analysis of DNA Content of Living and Fixed Cells: a Comparative Study Using Various Fixatives

The majority of studies dealing with DNA analyses are made on fixed cells. In this context, the efficiency as fixatives of ethanol, methanol, acetone, Carnoy, Boehm-Sprenger and aldehydes was determined using two different DNA fluorescent probes, Hoechst 33342 and propIDium iodIDe. The purpose of our study was to find the fixative that would provIDe the best results with respect to the following parameters: aggregates, cell size and granularity, and DNA staining analysis. Using murine fibroblasts, we found that 68% ethanol, 85% methanol and aldehydes dID not increase aggregate formation, whereas Carnoy, acetone or Boehm-Sprenger fixatives dID. The results show that aldehydes seem to alter cell size least. All fixatives induce an increase in cell granularity, which is very pronounced with alcohols, but aldehydes alter morphology less than alcohols. We observed that the fixatives giving the best resolution with Hoechst 33342 staining lead to a lower measurement variabili ty than with propIDium iodIDe staining. This study leads us to conclude that 68% ethanol and 85% methanol can be consIDered as appropriate fixatives for flow cytometry studies of DNA content.     

流式细胞术分析和分拣植物染色体

流式细胞术是当染色体、细胞核和细胞等颗粒随着流动的液体（水或缓冲液）通过一个测量点时,被探测器探测到,这样根据颗粒的物理和化学特征而将不同的颗粒分开并计数分拣的技术。流式细胞分析在人类基因组计划中发挥了重要作用,流式细胞技术的应用也适用于植物,目前这个技术应用范围包括流式核型分析,分拣纯化染色体,定位基因,构建文库等。文章综述了流式细胞术在植物基因组分析方面的研究进展。     

Flow Cytometric Analysis and Sorting of Heterodera glycines Eggs

A nondestructive technique was developed to characterize and separate eggs of soybean cyst nematode, Heterodera glycines, by developmental stage using flow cytometry. Eggs from cysts cultured on susceptible soybean roots were suspended in 0.1% xanthan gum or 59% sucrose and loaded into either a Coulter EPICS 752 or EPICS 753 flow cytometer. Eggs were analyzed and sorted according to forward angle and 90° light scatter, flow cytometric parameters that are relative measures of object size and granularity, respectively. Mature eggs containing vermiform juveniles were less granular and slightly larger than eggs in earlier stages of embryogeny, allowing for separation of mature eggs from immature eggs. The effectiveness of flow cytometric sorting was evaluated by comparing the developmental stages of subpopulations of unsorted and sorted eggs. Of a subpopulation of unsorted eggs, 62% contained vermiform juveniles, whereas 85 to 95% of sorted subpopulations of larger, less granular eggs contained vermiform juveniles. Suspending H. glycines eggs in 0.1% xanthan gum or 59% sucrose for flow cytometric analysis had no effect on subsequent egg hatch in vitro. This technique is an efficient and effective means to collect large, relatively homogeneous quantities of H. glycines eggs in early or late embryogeny, and would likely be useful for analyzing and sorting eggs of other nematode species for use in developmental, genetic, or physiological research, or for identification and collection of parasitized eggs.     

Spores of a New Microsporidan Species Parasitizing Molluscan Neurons

Synopsis.
A new species of Microsporida was observed in neurons of the marine mollusc Aplysia californica and the ultrastructure of its spores was investigated. The spores were ˜ 1.3 m long and had a thick 3-layered coat. An anchoring disc and polar aperture were present. The polaroplast occupied most of the anterior half of the spore. The vesicular portion included lengths of loosely packed lamellar structures as well as the usual vesicular profiles embedded in fibrillar material; it was entirely surrounded by tightly packed, electron-dense lamellae. The polar tube had 5 or 6 coils. In this stage of the life cycle (mature spores), the parasites did not appear to be disturbing the host cells. The organism was named Microsporidium aplysiae sp. n.     

A Flow Cytometric Analysis of the Nuclear 2C DNA Content in 17 Phaseolus Species (53 Genotypes)

The genus Phaseolus is characterized by a highly stable karyotype of 2n = 22. Despite this constancy, the size of the chromosomes varies, and crossing of species is possible only in a few cases. We determined the 2C nuclear DNA content of a number of Phaseolus species, cultivars and genotypes by flow cytometry, in order to realize the interspecific and intraspecific variation of the 2C value. The data range from 1.03 pg to 2.18 pg without any clear correlation to systematic relationships. The mean DNA values of wild and cultivated forms, as well as those of Andean and Mesoamerican genotypes, do not differ significantly. The variation is interpreted in terms of some nucleotypic adaptations. The data may be useful for molecular biological analyses, as well as for biotechnological and classical breeding programmes.     

Effects of Flow Cytometric Analysis on Morphology and Viability of Fragile Phytoplankton

We assessed damage done to especially delicate marine phytoplankton cells by passage through a Coulter Epics V flow cytometer. The cells did not distort or lyse after exposure to fluidics or to laser light to 1,000 mW. The cells did sustain damage evidenced by temporary growth rate depressions. The four clones tested eventually resumed control growth rates after growth lags to 48 h.     

Collection,Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.     

Trypanosoma cruzi: Flow Cytometric Analysis of Developmental Stage Differences in DNA

Flow cytometry and DNA binding-specific fluorescent reagents were used to compare the total DNA, G-C, and A-T content of the epimastigote and trypomastigote stages of Trypanosoma cruzi stocks. Significant total DNA differences of 2–12% between epimastigotes and trypomastigotes were found in three of six stocks studied. The epimastigote G-C content of five of six stocks was 4–8% higher than trypomastigotes, whereas the trypomastigote A-T content was 2.5–13% higher than the epimastigote A-T content. Although no obvious developmental stage association between total DNA and base composition was found, intrastage associations do exist. These observations were unaffected by nucleoprotein extraction implying that the observed differences between trypomastigotes and epimastigotes are not a consequence of nucleoprotein interference with DNA-binding fluorochromes. The nuclei and kinetoplasts of four T. cruzi stocks were isolated and analyzed. Developmental stage differences in nuclear and kinetoplast DNA are stock-dependent and base composition-dependent; both organelles contribute to the observed differences in DNA of intact cells. We found a nearly linear association between the percentage of total kinetoplast DNA, G-C, and A-T content. During metacyclogenesis, the G-C content decreases by approximately 7% as epimastigotes transform into metacyclic trypomastigotes. The decrease in G-C content precedes changes in morphology or in complement resistance. If the DNA changes are causally connected to developmental stage transformations in T. cruzi remains to be determined. However, our results could facilitate studies of the molecular genetic processes the parasite uses to successfully complete various phases of its life cycle and, consequently, the disease process it evokes.     

Flow Cytometric Analysis of the Different Ploidy Levels Observed in the Genus Ulex L. Faboideae-Genisteae in Brittany (France)

Abstract: A controversy arose in the last decade concerning the identity of U. gallii and the ploidy of gorses in Brittany. To determine, for a wide number of samples, the ploidy levels of indigenous or recently introduced gorses in Brittany, we used the flow cytometric method on DAPI fluorescent nuclear suspensions, together with size measurements of stomata and dry pollen grains. All plants belonged to the section Neowillkommia of the genus Ulex. They were found to form a polyploid series with 2x, 4x and 6x levels. All U. minor were diploid. All U. europaeus ssp. europaeus and all U. gallii, except a single specimen out of 65, were hexaploid. This specimen had cell dimensions very close to that of U. europaeus ssp. latebracteatus which is a tetraploid. Tissue-specific augmentation was found in Rhizobium-containing cells of root nodules and within the endosperm of immature seeds. The 2C DNA content of U. europaeus ssp. europaeus was estimated at 90% that of Pisum sativum, so weighing about 7.7 pg/2C. Neither U. gallii nor U. europaeus ssp. europaeus showed any difference in their ploidy level between erect and prostrate ecotypes. The taxonomic and nomenclatural consequences are discussed.     

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.     

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.     

Flow Cytometric Analysis of Circadian Changes in Platelet Activation Using Anti-Gmp-140 Monoclonal Antibody

The hemostatic activity of blood shows a circadian variation with a higher frequency of acute coronary events in the morning. The thrombotic tendency of blood is influenced by many factors, including platelets. Diurnal changes of in vivo platelet activation were investigated by whole blood flow cytometry in 10 young healthy male volunteers using anti-GMP-140 (anti-α-granule membrane protein 140 kD) monoclonal antibody at 3h intervals from 06:00 to 24:00. We also studied circulating platelet aggregates to investigate whether there exists a similarity between the results of these methods. Results of flow cytometric analysis indicate that there is an increase in platelet activation during the period from 06:00 to 09:00. Platelet activation then decreases gradually during the period from noon to midnight. These changes are accompanied by a similar trend in circulating platelet aggregates. This suggests that GMP-140 expression on platelets is synchronized with or followed by platelet aggregate formation in vivo, and increased platelet activation may predispose individuals to thrombosis at this time.     

Trypanosoma cruzi: Flow Cytometric Analysis. I. Analysis of Total DNA/Organism by Means of Mithramycin-Induced Fluorescence1,2

ABSTRACT. Total DNA/organism was determined by flow cytometry on stocks of 33 single-cell-isolate clones and one strain of mithramycin-stained Trypanosoma cruzi. Interstrain differences in mean total DNA/group of 34% and interclone differences in total DNA/organism of 41% were found. Microspectrofluorometric analyses of the trypomastigote stage of selected clones confirmed the flow cytometry data and indicated that the total DNA/organism differences were due to differences in DNA of both the nucleus and kinetoplast with the nucleus being the major contributing factor. These data imply that the potential for genetic diversity in T. cruzi may be very large.     

Flow Cytometric Analysis and Chromosome Sorting of Barley (Hordeum vulgare L.)

Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 M trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.     

Flow Cytometric Analysis of Rhodamine 123 Fluorescence during Modulation of the Membrane Potential in Plant Mitochondria

The fluorescent dye rhodamine 123, which selectively accumulates in mitochondria based on the membrane potential, was used with flow cytometry to evaluate variations in activity of mitochondria isolated from plant tissues. In the presence of succinate and ATP, potato (Solanum tuberosum L.) tuber mitochondrial activity was affected by metabolic inhibitors and compounds that modify the membrane potential. The more uniform the mitochondrial population, the higher the observed membrane potential. The reactive population corresponds to the proportion of intact mitochondria (94-97%) defined by classic methods. Changes in the light-scattering properties are more related to internal modifications affecting the inner membrane-matrix system of the mitochondria during metabolic modulation than to specific volume change or outer membrane surface modifications. We tested our approach using an Arum maculatum preparation that contains three different types of mitochondria and demonstrated the validity of the light-scatter measurements to distinguish the α, β, and [ill] mitochondria and to measure their ability to built up a membrane potential in the presence of succinate. These results demonstrate clearly that flow cytometric techniques using rhodamine 123 can be employed to study the activity in isolated plant mitochondria.     

Effects of Flow Cytometric Analysis and Cell Sorting on Photosynthetic Carbon Uptake by Phytoplankton in Cultures and from Natural Populations

Photosynthetic rates of phytoplankton were significantly lower after analysis by flow cytometry (FCM) than before. Exposure to the laser beam during the sorting process caused significant physiological damage. The cellular content of a radiolabel accumulated prior to FCM was not affected by FCM. Although it may not be possible to use FCM to preconcentrate cells for further physiological studies, samples may be incubated with stable or radioactive isotopes and then analyzed by FCM.