Fluorescence replication banding of frog chromosomes

To identify individual chromosomes of a frog karyotype by their fluorescence banding patterns, chromosomes were stained with actinomycin D and 4,6-diamidino-2-phenylindole (DAPI) after incorporation of BrdU during the late S-phase. The chromosomes of three Rana species which were selected for this study (R. ridibunda, R. lessonae and R. japonica) showed well-defined late replication bands. The fluorescence patterns obtained were the reverse of those produced by a 4Na-EDTA Giemsa-staining technique. Fluorescence patterns of the two water frog species (R. ridibunda and R. lessonae) were similar to each other, except for the different fluorescence of the centromeric heterochromatin, which gave extremely bright signals in R. ridibunda but no signal in R. lessonae. Experiments also showed differences between the fluorescence patterns of R. lessonae chromosome 13 in the Italian and Luxembourgian populations. These results sho w that the fluorescence replication banding using actinomycin D and DAPI is very effective in identifying individual frog chromosomes and detecting their structural changes. Received 7 June 1996; received after revision 23 July 1996; accepted 21 August 1996     

A cell-marking technique for a cellular slime-mold

Summary A simple technique for marking cells of the cellular slime moldDictyostelium discoideum has been developed, using the phagocytic action of the cells. TheD. discoideum are fed onE. coli stained with neutral red and clearly red colored amoebae are obtained.     

Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342

Dictyostelium discoideum cells are highly resis tant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size >21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism. Received 14 November 1997; received after revision 16 March 1998; accepted 16 March 1998     

Distribution patterns of mammalian-like peptide immunoreactive cells in the midgut ofAeshna cyanea (Insecta,Odonata)

Summary The distribution of cells reacting with antisera to cholecystokinin, substance P, gonadoliberin, methionine-enkephalin, and vasoactive intestinal peptide, demonstrated by the indirect immunoperoxidase method, was studied along the entire midgut of an insect,Aeshna cyanea. For each antiserum, the number of reacting cells increased from the middle part to the end of the midgut. Only a few cells reacted to somatoliberin, leucin-enkephalin and somatostatin antisera. In the connective sheath surrounding the midgut epithelium, nerve fibers were stained by antisera to serotonin, somatostatin, cholecystokinin, vasoactive intestinal peptide and methionine-enkephalin.     

Low pH in fungal bud initials

Summary Quenching of fluorescence of the pH probe, 4-methylesculetin, in bud initials ofAllomyces hyphae and yeast (Saccharomyces) vegetative cells confirms the cytoplasmic acidity (pH not more than 5) in such amitochondrial structures.     

Effects of mistletoe (Viscum album L.) extracts on cultured tumor cells

Summary Bacterially fermented mistletoe preparations (BFMP) were tested on rat hepatoma tissue culture (HTC) cells and human leukemia Molt 4 cells. A dose-dependent inhibition of the growth rate of the cells was observed. For both cell lines, cytostatic concentrations, expressed in weight of fresh plant, were 0.5 mg/ml culture medium for oak BFMP and 1 mg/ml for apple tree BFMP. However, the action of the two preparations was markedly different on each cell line. Non-viable HTC cells were not stained by trypan blue while non-viable Molt 4 cells were fully colored by this reagent. A lysis of cellular membranes of HTC cells was observed by electron microscopy. Furthermore, oak BFMP inhibited the growth of virus transformed 3T3-SV40 cells more than that of non-transformed 3T3 cells. In contrast to BFMP, non-fermented extracts and a purified mistletoe lectin showed a greater inhibition of the growth of Molt 4 cells than of HTC cells. Samples withdrawn at different times during fermentation gradually lost their inhibitory effect on the growth of Molt 4 cells while their action on HTC cells increased up to the 4th day of fermentation. These results are discussed in relation to the cytotoxic substances of mistletoe already characterized.The bacterially fermented mistletoe preparations, named BFMP in the text, were obtained from the Hiscia Institute, CH-4144 Arlesheim, Switzerland, under the name of Iscador. For oak BFMP, mistletoe was fromQuercus petraea Liebl. andQuercus robur L.; for apple tree BFMP fromMalus domestica Borkh.     

Altered microviscosity of in vivo lipid-manipulated membranes inTetrahymena pyriformis: A fluorescence study

Summary By determination using fluorescence polarization measurements with 1,6-diphenyl 1,3,5-hexatriene, ergosterolreplaced pellicle and microsome membranes ofTetrahymena cells become less fluid, whereas those membranes from chimyl alcohol-fed cells are more fluid, when compared with the control native membranes.Acknowledgments. We wish to express thanks to Central Scientific Commerce, Inc., Tokyo for permission to use an Elscint Microviscosimeter MV-Ia. This work was in part supported by grant from the Ministry of Education.     

Drosophila unconventional myosin VI is involved in intra- and intercellular transport during oogenesis

During mid-oogenesis of Drosophila, cyto plasmic particles are transported within the nurse cells and through ring canals (cytoplasmic bridges) into the oocyte by means of a microfilament-dependent mecha nism. Video-intensified fluorescence timelapse mi croscopy, in combination with microinjections of antibodies directed against Drosophila 95F myosin, have revealed that this unconventional myosin of class VI is involved in the transport processes. The results indicate that certain cytoplasmic particles in the nurse cells move along microfilaments due to their direct association with myosin VI motors. Additional myosin- VI molecules located at the rim of the ring canals seem to be involved in particle transport into the oocyte. Microinjected mitochondria-specific dyes have revealed that some of these particles are mitochondria. Received 3 April 1997; received after revision 5 May 1997; accepted 27 May 1997     

Evolution of the thermotropic properties of large unilamellar vesicles (LUV)

Summary The evolution of the thermotropic properties of large unilamellar vesicles (LUV) made by the reverse-phase evaporation technique has been studied by differential scanning calorimetry (DSC) and by fluorescence polarization of the diphenylhexatriene probe inserted in the lipid phase. Lipid fluidity and transition temperatures of DL-a-dimyristoyl-and DL-a-dipalmitoyl-phosphatidylcholine vesicles were practically not modified at room temperature (19–20°C), even after several days. Because a better knowledge of the physico-chemical properties of LUV seems essential for its use as a model membrane or as a carrier of exogenous material into cells, we compare it with the stability of the widely used multilamellar (MLV) and sonicated unilamellar vesicles (SUV).     

Analysis of <Emphasis Type="Italic">Dictyostelium</Emphasis> TACC reveals differential interactions with CP224 and unusual dynamics of <Emphasis Type="Italic">Dictyostelium</Emphasis> microtubules

We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dynamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-α-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers.