Non-random position of the A-T rich DNA sequences in early embryos of Drosophila virilis

Examination of early embryos of Drosophila virilis by light and electron microscopy has shown that the A-T rich satellite DNA sequences have a non-random distribution within the nuclei. As observed by 33258 Hoechst staining and fluorescent microscopy, these sequences are consistently found to be located on the sides of the nuclei nearest to the vitelline membrane. This arrangement of the A-T rich sequences has been observed from the syncytial balstoderm stage into the gastrula stage where, in each nucleus, the satellite DNA sequences remain at a point nearest the topological outside of the organism.     

Cell age-related differences in the interaction of a potential-sensitive fluorescent dye with nuclear envelopes of Acetabularia mediterranea

Abstract. To study whether an electrical potential difference exists across the nuclear envelope or inner nuclear membrane of plant cells, the authors have used an optical probe of membrane potential, the cationic fluorescent dye, DiOC₆(3) (MW = 572.5). This dye was microinjected into the nucleoplasm of isolated Acetabularia nuclei (which are still surrounded by a thin layer of cytoplasm) and its subnuclear localization visualized by fluorescence microscopy. Striking differences, which seemed to be correlated with the developmental stage of the isolated nucleus, were observed. In nuclei isolated from cells at the stage of early cap stage formation, the dye was restricted to the nuclear envelope. In nuclei isolated from cells with intermediate or fully developed caps, there was increased nucleoplasmic staining, and the staining of the envelope was frequently diminished or abolished. In all nuclei, the dye remained within the nucleus after injection. Cytoplasmic staining was only observed when nuclei isolated from cells at the stage of early cap formation were incubated in a hyper- or hypo-tonic medium. Various ionophores, injected before the dye into the nucleoplasm, had no effect on the subsequent nuclear localization of DiOC₆(3), although they did rapidly induce nucleolar condensation in nuclei isolated from cells at the stage of early cap formation. The results suggested that the electrical properties of Acetabularia nuclear envelopes or inner nuclear membranes change during cell maturation. Furthermore, the retention of the dye in the nucleoplasm under isotonic conditions indicated that the nuclear pores were not open channels for molecules of this size.     

Generation of a transgenic medaka (Oryzias latipes) strain for visualization of nuclear dynamics in early developmental stages

In this study, we verified nuclear transport activity of an artificial nuclear localization signal (aNLS) in medaka fish (Oryzias latipes). We generated a transgenic medaka strain expresses the aNLS tagged enhanced green fluorescent protein (EGFP) driven by a medaka beta‐actin promoter. The aNLS‐EGFP was accumulated in the nuclei of somatic tissues and yolk nuclei of oocytes, but undetectable in the spermatozoa. The fluorescent signal was observed from immediately after fertilization by a maternal contribution. Furthermore, male and female pronuclei were visualized in fertilized eggs, and nuclear dynamics of pronuclear fusion and subsequent cleavage were captured by time‐lapse imaging. In contrast, SV40NLS exhibited no activity of nuclear transport in early embryos. In conclusion, the aNLS possesses a strong nuclear localization activity and is a useful probe for fluorescent observation of the pronuclei and nuclei in early developmental stage of medaka.     

Cytological studies of plasma albumin in the rat liver

Summary The presence of albumin in the parenchymal cells of the rat liver is demonstrated using the fluorescent antibody method. It is evident that there is no immunological difference between liver and blood albumin. The liver section is not uniformly stained by the fluorescent antibody and this may be due to the uneven distribution of albumin in the liver. A small percentage of the parenchymal cells presumably containing albumin are markedly fluorescent. The nuclei of these cells are not fluorescent implying the absence of albumin and this is consistent with the fact that nuclei are not involved in synthesis of albumin. The scattered distribution of brightly fluorescent cells confirms earlier observations that all parenchymal cells are not functionally alike, although they appear homogenous histologically.     

HeLa细胞核和核骨架含有肌动蛋白

提取HeLa细胞核并制备核骨架标本,以抗肌动蛋白抗体为探针,采用SDS－PAGE、免疫荧光和免疫印迹等方法,对HeLa细胞细胞核和核骨架中的肌动蛋白进行了研究,并用鬼笔环肽荧光染色方法研究了其中的F－肌动蛋白。在荧光显微镜下观察到：代表肌动蛋白的特异性荧光分布在细胞核和核骨架中,说明肌支蛋白是细胞核和核骨架的固有成分;代表F－肌动蛋白的特异性荧光存在于细胞和核骨架中,说明细胞核和核骨架含有F－肌动蛋白。免疫印迹结果进一步肯定了细胞核和核骨架中肌动蛋白的存在。     

Studies on sex-organ development. Changes in nuclear and chromatin composition and genomic activity during spermatogenesis in the maturing rooster testis.

We developed a technique to separate nuclei of rooster testis by centrifugation through a discontinuous sucrose density gradient and by sedimentation at unit gravity. Four different major fractions obtained from testicular nuclei and one from the vas deferens were characterized according to their velocity of sedimentation, morphology and DNA content. The ratios (w/w) of basic proteins, non-histone proteins and RNA to DNA decreased during spermiogenesis both in nuclei and chromatin. Changes in the electrophoretic patterns of histones and non-histone proteins were detected especially in the elongated spermatids. The lack of uptake of [3H]uridine in elongating and elongated spermatids and in spermatozoa was demonstrated by radioautography and by the detection of labelled RNA extracted from different fractions of nuclei. Template activity for RNA synthesis and the binding of actinomycin D by testicular nuclei reached a peak in the elongated spermatid stage, when the histones are replaced by the protamine.     

DNA flow cytometric analysis of mouse seminiferous epithelium

In order to provide a basis for quantitative studies of murine spermatogenesis, we performed a DNA flow cytometric analysis on the mouse seminiferous tubules isolated at defined stages of the epithelial cycle by transillumination-assisted microdissection. Accurate stage identification was performed by examining spermatids in the adjacent tubule segments by phase-contrast microscopy. For flow cytometry, suspension of nuclei of spermatogenic cells was obtained by detergent treatment of isolated seminiferous tubules, and fresh samples were stained with propidium iodide. DNA histograms of the 12 stages of the mouse seminiferous epithelial cycle varied in a stage-specific manner. DNA histograms of stages I-VIII of the cycle were characterized by a hypofluorescent haploid peak, the location of which changed with the decreasing DNA dye (propidium iodide)-binding capacity of elongated spermatids. The absence of the hypohaploid peak and the high ratio of the cells with 4C amount of DNA to the cells with 1C amount of DNA characterized stages IX-XI of the cycle. Stage XII showed a high 2C peak, owing to a large population of secondary spermatocytes arisen from the first meiotic division. By using fluorescent beads as an internal volume standard cell numbers in defined stages were determined. These data provide a basis for quantitative studies of mouse spermatogenesis.     

Tumor Cells Genetically Labeled with GFP in the Nucleus and RFP in the Cytoplasm for Imaging Cellular Dynamics

Dual-color fluorescent cells with one color fluorescent protein in the nucleus and another color fluorescent protein in the cytoplasm were genetically engineered. The dual-color cancer cells enable real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed in the cytoplasm of a series of human and rodent cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Nuclear GFP expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression enabled visualization of nuclear-cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Using the Olympus OV100 Whole-Mouse Imaging System, total sub-cellular dynamics can be visualized in the living dual-color cells in real time in the live mouse after cell injection. Highly elongated cancer cells and nuclei in narrow capillaries were visualized where both the nuclei and cytoplasm deform. Both cytoplasm and nuclei were visualized to undergo extreme deformation during extravasation with cytoplasmic processing exiting vessels first and nuclei following along these processes. The dual-color cells described here thus enable the sub-cellular dynamics of cancer cell trafficking to be imaged in the living animal.     

Programmed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx mori

In the present study, we describe the features of programmed cell death of the ovarian nurse cells occurring during vitellogenesis of the silkmoth Bombyx mori. At developmental stage 5, the nurse cells occupy one-half of the follicular volume and obtain a rather spherical shape, while the nurse cell nuclei appear large and elongated, forming impressive projections. At the following stage, stage 6, the nurse cells decrease in size and their shape becomes elliptic. The nuclei remain elongated, being also characterized by large lobes. The lobes of the ramified nurse cell nuclei seem to retain the nucleus in the center of the cell during the dumping of the nurse cell cytoplasm into the growing oocyte. At stage 7, membrane enclosed vacuoles can be easily detected into the nurse cells cytoplasm. Ultrastructural analysis and fluorescent microscopy using mono-dansyl-cadaverine staining of these vacuoles also reveal that they represent autolysosomes. Caspase activity is detected during stage 7, as it is demonstrated by using the Red-VAD-FMK staining reagent. At developmental stages 8 and 9, the nurse cells exhibit chromatin condensation, DNA fragmentation and caspase activity. Finally, during the following stage 10, the nuclear remnants are assembled into apoptotic vesicles, which, after being phagocytosed, are observed in the cytoplasm of adjacent follicle cells. We propose that apoptosis and autophagy operate synergistically during vitellogenesis of B. mori, in order to achieve an efficient and rapid clearance of the degenerated nurse cell cluster.     

Full-term development of rat after transfer of nuclei from two-cell stage embryos

Cloning technology would allow targeted genetic alterations in the rat, a species which is yet unaccessible for such studies due to the lack of germline-competent embryonic stem cells. The present study was performed to examine the developmental ability of reconstructed rat embryos after transfer of nuclei from early preimplantation stages. We observed that single blastomeres from two-cell embryos and zygotes reconstructed by pronuclei exchange can develop in vitro until morula/blastocyst stage. When karyoplasts from blastomeres were used for the reconstruction of embryos, highest in vitro cleavage rates were obtained with nuclei in an early phase of the cell cycle transferred into enucleated preactivated oocytes or zygotes. However, further in vitro development of reconstructed embryos produced from blastomere nuclei was arrested at early cleavage stages under all conditions tested in this study. In contrast, immediate transfer to foster mothers of reconstructed embryos with nuclei from two-cell embryos at an early stage of the cell cycle in preactivated enucleated oocytes resulted in live newborn rats, with a general efficiency of 0.4%-2.2%. The genetic origin of the cloned offspring was verified by using donor nuclei from embryos of Black Hooded Wistar rats and transgenic rats carrying an ubiquitously expressed green fluorescent protein transgene. Thus, we report for the first time the production of live cloned rats using nuclei from two-cell embryos.     

Cell function in the ovary of drosophila

Summary The relative DNA content of the ovarian nurse nuclei of Drosophila melanogaster has been measured by high-resolution autoradiography of DNA uniformly labelled with adenine-8-¹⁴C.The various nurse nuclei show a defined pattern of DNA classes. The posterior nuclei, i. e. those nearest to the oocyte, achieve eight reduplications of DNA by stages 8–9, thus reaching 512n, and all have lost some DNA by stage 10. The nuclei in the middle of the chamber achieve seven reduplications of DNA by stage 9, thus reaching 256n, and though there is loss of DNA in the majority of these nuclei at stage 10 some of them might enter a new reduplication cycle. The anterior nuclei, i. e. those more distant from the oocyte, achieve more than seven reduplications by stage 10 and show no loss of DNA.After stage 6 of the ovarian chambers the pattern of DNA enrichment and later degradation is clearly polarized in that there is a posterioranterior gradient for the level of ploidy, the order in time in which this is attained, and the loss of DNA. The dominant end of the gradient is towards the developing oocyte.The measured nuclear volume where DNA is present is well correlated with ploidy till stage 9. Compared with earlier stages, at stage 10 DNA shares less in the nuclear contents than other materials. The nuclear volume when calculated as a sphere is a gross overestimation, except for the earliest stages.Various possibilities likely to bring about differences in the amount of DNA among nurse nuclei within and between chambers are discussed.Research worker of the British Empire Cancer Campaign.     

A rapid method for flow cytometric cell counting and cell cycle analysis from monolayer cultures of tumor cells.

A rapid, simple, and reliable flow cytometric method using the histochemical fluorescent stain Hoechst 33342 in presence of the non-ionic detergent Triton X-100 has been reported. The processing of melanoma cell cultures to get nuclei stained with the fluorescent dye was accomplished in one step and within an hour permitted concurrent flow cytometric measurement of cell density and cell cycle analysis. The preparation is stable for more than three weeks at room temperature for flow cytometry. The histograms are reproducible and exhibit a coefficient of variation of less than 2.5% (G1 peak). The cell density measurements varied within +/- 5% limits.     

利用荧光蛋白标记西瓜枯萎病菌的过氧化物酶体及细胞核

西瓜枯萎病是一种世界范围的西瓜毁灭性病害,其病原菌为尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum,FON)。研究病原菌生长发育和侵染的机制是解决病害的根本途径。利用荧光蛋白对细胞或细胞器进行标记,是病原菌研究中的重要方法。该研究利用绿色荧光蛋白和红色荧光蛋白对FON的细胞核和过氧化物酶体进行了荧光标记。通过农杆菌介导转化(Agrobacterium tumefaciens-mediated transformation,AtMT),该文将3种不同的荧光定位载体分别导入FON,获得了细胞核红色荧光标记的转化子(潮霉素抗性,含mCherry-H2B融合蛋白),以及过氧化物酶体绿色(潮霉素抗性,含GFP-PTS1融合蛋白)和红色(潮霉素抗性,含DsRED-PTS1融合蛋白)荧光标记的转化子各1种。在标记细胞核的菌株中,菌丝、孢子都可见明亮、圆形的红色荧光点,荧光点与DAPI染色标记的细胞核区域完全重合。在过氧化物酶体标记的菌株中,菌丝、孢子中可见明亮的红色或绿色荧光成小点状分布,符合过氧化物酶体的分布特征,而且在脂类物质诱导的条件下,荧光点的数量明显增加。此外,该文还利用细胞壁荧光染色剂卡氏白对3种荧光蛋白标记菌株进行染色。结果显示,卡氏白染色产生的蓝色荧光与红、绿荧光蛋白的荧光在FON中互不干扰。转化子继代培养和初步分析表明,其表型与野生型无差异,菌株继代后荧光表达稳定、定位明显。该结果为进一步研究FON细胞器动态、生长发育与致病分子机制提供了方法和工具。     

Tracking nucleolar dynamics with GFP-Nopp140 during Drosophila oogenesis and embryogenesis

We expressed two green fluorescent protein (GFP)-tagged Nopp140 isoforms in transgenic Drosophila melanogaster to study nucleolar dynamics during oogenesis and early embryogenesis. Specifically, we wanted to test whether the quiescent oocyte nucleus stored maternal Nopp140 and then to determine precisely when nucleoli formed during embryogenesis. During oogenesis nurse cell nucleoli accumulated GFP-Nopp140 gradually such that posterior nurse cell nucleoli in egg chambers at stage 10 were usually brighter than the more anterior nurse cell nucleoli. Nucleoli within apoptotic nurse cells disassembled in stages 12 and 13, but not all GFP-Nopp140 entered the oocyte through inter-connecting cytoplasmic bridges. Oocytes, on the other hand, lost their nucleoli by stage 3, but GFP-Nopp140 gradually accumulated in oocyte nuclei during stages 8–13. Most oocyte nuclei at stage 10 stored GFP-Nopp140 uniformly, but many stage 10 oocytes accumulated GFP-Nopp140 in presumed endobodies or in multiple smaller spheres. All oocyte nuclei at stages 11-12 were uniformly labeled, and GFP-Nopp140 diffused to the cytoplasm upon nuclear disassembly in stage 13. GFP-Nopp140 reappeared during embryogenesis; initial nucleologenesis occurred in peripheral somatic nuclei during embryonic stage 13, one stage earlier than reported previously. These GFP-Nopp140-containing foci disassembled at the 13th syncytial mitosis, and a second nucleologenesis occurred in early stage 14. The resulting nucleoli occupied nuclear regions closest to the periphery of the embryos. Pole cells contained GFP-Nopp140 during the syncytial embryonic stages, but their nucleologenesis started at gastrulation. This work was supported by the National Science Foundation (grant MCB-0234245). O'Keith Dellafosse was supported by the Louisiana Alliance for Minority Participation (LAMP).     

Microfluorometric estimates of proteins associated with murine hepatocyte and thymocyte nuclei, residual structures, and nuclear matrix derivatives

Isolated diploid hepatocyte and thymocyte nuclei and their derivatives ("residual structures" and nuclear matrices, as defined by Kaufmann et al. 1981) were evaluated by microfluorometry following reaction with the following fluorochromes: brilliant sulfaflavine (BSF) used at pH 2.8 for the demonstration of total protein; acridine orange (AO) used at pH 9.0 to reveal acidic groups of proteins; and 3-(4-maleimidylphenyl)-7-diethylamino-4-methylcoumarin (CPM) used under conditions required to demonstrate the sum of sulfhydryl (SH) and disulfide (SS) groups of proteins. The results suggested that the proteins reacting with AO and CPM differed from each other and from those revealed by fluorochroming with BSF. In every comparison, hepatocyte nuclei and their derivatives were more fluorescent than the respective populations of thymocyte nuclei and their derivatives. In material fluorochromed with BSF and AO, nuclear matrices were less fluorescent than residual structures, which, in turn, were less fluorescent than intact nuclei. In contrast, nuclear matrices fluorochromed with CPM were less fluorescent than intact nuclei but more fluorescent (paradoxically) than residual structures. The ratios of the total fluorescence values of hepatocyte and thymocyte nuclei fluorochromed with BSF changed significantly during extractions required to produce residual structures and nuclear matrices, while comparable ratios in material fluorochromed with AO or CPM did not change significantly. Comparisons of the ratios of the fluorescence values of intact nuclei and their derivatives in a variety of combinations yielded complex and variable results.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Monoaminergic Paraventricular Organ in the Teleost Ictalurus nebulosus LeSueur,with Special Reference to Its Vascularization

Specific, fluorescent, subependymal perikarya were found in the pars anterior of the paraventricular organ (PVOpa), in the nucleus recessus lateralis (NRL) and in the nucleus recessus posterioris (NRP). No fluorescent perikarya were present in the nucleus lateralis tuberis (NLT). Fluorescent nerve tracts connect the PVOpa and the NRL with the NRP, and interconnect the paired NRP. The nucleus preopticus (NPO) and the NLT receive a large input of aminergic nerve fibers. The monoaminergic nuclei are well vascularized, and their vascular plexes seem to be connected. A capillary plexus is situated dorsal to the NRP and exhibits no contact with the pituitary. It is surrounded by the prominent fluorescent tracts connecting the aminergic nuclei.     

Fluorescence microscopy of the catecholamine-containing neurons of the hypothalamohypophyseal system

Summary The hypothalamohypophyseal system of the mouse, rat, guinea-pig, cat, dog and monkey (Macaca mulatta) was studied with the fluorescence method for catecholamine-containing neurons developed by Falck et al. (1962). The fluorescent fibers are prominent in the external layer and around the primary portal plexus of the infundibulum and in the peripheral region of the neural lobe of these animals, particulary on the external surface and surrounding the primary capillary loops. These fluorescent fibers are connected with fluorescent cells in the arcuate nuclei, and this connection coincides with the tuberohypophyseal system. The neurons of this system have a particular affinity for dopamine, possibly due to their own content of dopamine. In the supraoptic and paraventricular nuclei, no fluorescent cells were found. In the pars intermedia, we also found catecholamine-containing fibers.The presence of catecholamine-containing fibers in the adeno- and neurohypophysis are considered in relation to other data derived from fluorescence and electron microscopy.     

Polyploidization of nuclei in the yolk syncytial layer of the embryo of the medaka, Oryzias latipes, after the halt of mitosis

It has been reported that nuclei repeat parasynchronous mitosis four or five times in the yolk syncytial layer (YSL) of the embryo of the medaka, Oryzias latipes , during the blastula stage and that no mitosis occurs in the YSL after the gastrula stage. The present investigation demonstrated the size of nuclei and the number of nucleoli and their staining properties with DNA binding dye. The results indicate that the YSL nuclei actively transcribe RNA and that their DNA content is greater than that of somatic nuclei. The onset and subsequent time course of polyploidization were examined in embryos stained with 4',6-diamidino-2-phenylindole (DAPI) by epifluorescence microspectrophotometry from the cessation of mitosis through hatching. Embryos included YSL nuclei whose DNA content spanned from diploid (2C), tetraploid (4C) to octaploid (8C) at the end of the late blastula stage. The last two populations are produced probably by their early cessation of mitosis and the subsequent duplication of DNA without mitosis or by endoreduplication. The frequency distribution of the DNA content examined during epiboly of the blastoderm suggests that each population is duplicated again until the beginning of the gastrula stage and then once more until the end of epiboly. Eventually these nuclei include polyploid DNA between 8C and 64C or more during later embryonic development.     

Microscopical and spectroscopic studies on the fluorescence of a daunomycin-aluminum complex.

In this study, the spectroscopic features and microscopical applications of the fluorescent daunomycin-Al3+ complex have been analyzed. In the presence of Al3+, the absorption spectrum of daunomycin showed a deep bathochromic shift and new peaks at 529 and 566 nm, whereas the fluorescence emission was considerably modified. The emission of daunomycin alone (peak at 560 nm under optimal excitation at 470 nm) decreased continuously from 0.5 to 24h after addition of Al3+ ions, and a new emission peak appeared at 580 nm (optimal excitation at 530 nm). Under the fluorescence microscope using green exciting light, nuclei from chicken blood smears and paraffin sections of rat embryos stained with daunomycin showed a weak emission, which greatly increased after treatment with Al3+ ions. The bright and stable fluorescence of chromatin DNA induced by daunomycin-Al3+ could be a valuable labelling method in fluorescence microscopy and DNA cytochemistry.