Observation of polyphosphate bodies and DNA during the cell division cycle of Synechococcus elongatus PCC 7942

Although most cyanobacterial cells contain prominent polyphosphate bodies in the central cytoplasmic area enclosed by the peripheral thylakoid membranes, their roles are not fully understood. Storing phosphate for nucleotide production might be one of their important roles in survival of the cells. As a step towards identifying a possible contribution of the polyphosphate bodies to DNA synthesis, the relationship between polyphosphate bodies and DNA throughout cell division cycle of Synechococcus elongatus PCC 7942 cells cultured under light/dark cycles was investigated with light and electron microscopy. During the dark period, the average size of polyphosphate bodies increased gradually without significant change in their number and distribution. However, during the light period, the number of polyphosphate bodies increased, while the size of each polyphosphate body decreased and cells elongated until the end of the light period, when most cells divided. The ratio of the content of polyphosphate bodies to cell length increased gradually during the dark period and decreased during the light period. Hoechst 33342‐stained DNA appeared diffuse during the dark period, but in the light period it became condensed and eventually formed a wavy, rope‐like structure prior to cell division. Close association between fibres containing DNA and polyphosphate bodies was demonstrated by TEM using DNA‐specific staining and BrdU labelling. These regular coordinated changes of polyphosphate bodies and DNA shape during the cell division cycle, together with their intimate interaction, imply a role of polyphosphate bodies in supplying material for DNA.     

Composition of nuclear dense bodies and nucleolus-associated bodies in interphase nuclei of the unicellular green alga Chlamydomonas reinhardtii

Summary— The interphase nucleus of the green alga, Chlamydomonas reinhardtii, displayed two types of bodies some of them, the dense bodies, lying apparently free in the nucleoplasm while the others were attached to the nucleolus and were, therefore, referred to as nucleolus-associated bodies (NABs). The presence of DNA, RNA and histones in dense bodies was investigated by means of post-embedding immunocytochemistry and cytochemistry using a monoclonal antibody to single and double stranded DNA, a polyclonal antibody to rye H3 histones and RNase A-gold complexes. The dense bodies were shown to contain significant amounts of RNA but neither DNA nor histones were detected; their composition was thus similar to that of the dense bodies described in higher plant cells. We propose that dense bodies might be implicated in the assembly of the 25 to 45 nm granules observed throughout the nucleoplasm of Chalamydomonas interphase nuclei. The composition of NABs was found to be distinct from that of the dense bodies since they were labeled by the antibody to DNA, specially in cryofixed and cryosubstituted specimens. The presence of DNA in NABs together with their intimate association to the nucleolus suggest that they may correspond to specific segments of chromosomes.     

The cell cycle of Bacillus subtilis as studied by electron microscopy

Bacillus subtilis strain Marburg was grown exponentially with a doubling time of 65 min. To follow the time course of various cell cycle events, cells were collected by agar filtration and were then classified according to length. The DNA replication cycle was determined by a quantitative analysis of radioautograms of tritiated thymidine pulse labeled cells. The DNA replication period was found to be 45 min. This period is preceded and followed by periods without DNA synthesis of about 10 min.The morphology and segregation of nucleoplasmic bodies was studied in thin sections. B. subtilis contains two sets of genomes. DNA replication and DNA segregation seem to go hand in hand and DNA segregation is completed shortly after termination of DNA replication.Cell division and cell separation were investigated in whole mount preparations (agar filtration) and in thin sections. Cell division starts about 20 min after cell birth; cell separation starts at about 45 min and before completion of the septum.     

Recombinant Galectins of <Emphasis Type="Italic">Haemonchus contortus</Emphasis> Parasite Induces Apoptosis in the Peripheral Blood Lymphocytes of Goat

The effect of Haemonchus contortus galectin peptides rHco-gal-m/f to induce apoptosis in the peripheral blood lymphocytes (PBLCs) of goats was investigated. Analysis of apoptosis was carried out with agarose gel electrophoresis, flow cytometry and transmission electron microscopy. The results indicated that there were visible apoptosis bodies and typical DNA ladders by genomic DNA fragmentation. The quantitative analysis of apoptosis by flow cytometry indicated that rHco-gal-m/f peptides induced apoptosis was time and dose dependent. Ultrastructural studies of the PBLCs revealed that a large number of apoptotic cells were present in galectin-treated cells, which had the typical morphologic changes of apoptosis such as reduction of the cytoplasmic volume, loss of cell surface microvilli, chromatin condensation and fragmentation of the apoptotic cells into small apoptotic bodies.     

Isolation and characterization ofAnaplasma marginale DNA

Two procedures were used to isolateAnaplasma marginale bodies from bovine erythrocytes. DNA extracted from bodies prepared by the second method was free of any detectable bovine DNA contamination.Anaplasma marginale DNA was analyzed by agarose gel electrophoresis of endonuclease restriction fragments and by reassociation kinetics. Genome size was estimated to be 340 kb. Base composition of the DNA was 33 mol% guanine+ cytosine (G+C), determined from its thermal denaturation temperature.Anaplasma marginale has a very small genome compared with that of other bacteria and has a low G+C content. It is proposed thatA. marginale may be a close, but degenerate, relative of the rickettsiae.     

A novel single‐cell method provides direct evidence of persistent DNA damage in senescent cells and aged mammalian tissues

The DNA damage response (DDR) arrests cell cycle progression until DNA lesions, like DNA double‐strand breaks (DSBs), are repaired. The presence of DSBs in cells is usually detected by indirect techniques that rely on the accumulation of proteins at DSBs, as part of the DDR. Such detection may be biased, as some factors and their modifications may not reflect physical DNA damage. The dependency on DDR markers of DSB detection tools has left questions unanswered. In particular, it is known that senescent cells display persistent DDR foci, that we and others have proposed to be persistent DSBs, resistant to endogenous DNA repair activities. Others have proposed that these peculiar DDR foci might not be sites of damaged DNA per se but instead stable chromatin modifications, termed DNA‐SCARS. Here, we developed a method, named ‘DNA damage in situ ligation followed by proximity ligation assay’ (DI‐PLA) for the detection and imaging of DSBs in cells. DI‐PLA is based on the capture of free DNA ends in fixed cells in situ, by ligation to biotinylated double‐stranded DNA oligonucleotides, which are next recognized by antibiotin anti‐bodies. Detection is enhanced by PLA with a partner DDR marker at the DSB. We validated DI‐PLA by demonstrating its ability to detect DSBs induced by various genotoxic insults in cultured cells and tissues. Most importantly, by DI‐PLA, we demonstrated that both senescent cells in culture and tissues from aged mammals retain true unrepaired DSBs associated with DDR markers.     

CYTOCHEMICAL STUDIES ON PROCHLOROPHYTES: LOCALIZATION OF DNA AND RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE-OXYGENASE1

We studied the distribution of the DNA-containing region and the ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCo) content of polyhedral bodies in three different prochlorophyte cell types in a search for broad evolutionary affinities of these chlorophyll b-containing prokaryotes. DNA was localized by DAPI staining and electron microscopy utilizing monoclonal anti-DNA antibody 2C-10 plus a secondary antibody labeled with colloidal gold. Antibodies against the large RuBisCo subunit from a higher plant raised in rabbits were used to localize RuBisCo in polyhedral bodies. We studied Prochloron Lewin cells from two different didemnid ascidian hosts (Lissoclinum patella and Didemnum molle) collected in Palau, West Caroline Islands, and cells of Prochlorothrix hollandica Burger-Wiersma, Stal, and Mur grown in laboratory culture. Cells of the blue-green alga Anabaena 7120 were studied for comparison. The DNA distribution was markedly different in the two Prochloron cell types. The thylakoids in cells from L. patella were concentrically arranged around a large central vacuole; the DNA-containing stromal areas appeared in thin sections as a concentric arcs between the thylakoid stacks. The central vacuole was lacking in cells from D. molle, and the thylakoid stacks and strands of DNA-containing stroma showed a more haphazard arrangement. In the filamentous Prochlorothrix the DNA-containing stroma was largely limited to a central nucleoid structure running the length of the cell. Although the DNA arrangements in Prochloron might be considered “chloroplast-like” since DNA-containing stroma is distributed, as in chloroplasts, in scattered sites among photosynthetic membranes, this is not so in Prochlorothrix, where there is an axial nucleoid, as in many filamentous cyanobacteria. Our anti-RuBisCo antibodies were selectively bound to the polyhedral bodies of all three cell types, indicating that Prochloron and Prochlorothrix, like many other autotrophic prokaryotes, possess typical carboxysomes.     

WIDESPREAD PHAGOCYTOSIS OF CILIATES AND OTHER PROTISTS BY MARINE MIXOTROPHIC AND HETEROTROPHIC THECATE DINOFLAGELLATES1

An electron microscopic examination of large amorphous inclusions located in a variety of photosynthetic thecate dinoflagellates (Alexandrium ostenfeldii (Paulsen) Balech et Tangen, Gonyaulax diegensis Kofoid, Scrippsiella sp., Ceratium longipes (Bailey) Gran, and Prorocentrum micans Ehrenberg) and a nonphotosynthetic thecate species (Amylax sp.) revealed each inclusion to be a food vacuole, the majority of which were ingested ciliate prey. Recognizable features of these ciliates included linear arrays of basal bodies and cilia consistent with oligotrich polykinetid structure, characteristic macronuclei, chloroplasts (evidently kleptoplastids), cup-shaped starch plates, and cylindrical extrusomes. Three species contained (apparent) nonciliate prey: Scrippsiella sp., whose food vacuoles consistently contained unusual and complex extrusome-like cylindrical bodies having a distinctive six-lobed, multilayered structure; P. micans, which contained an unidentified encysted cell; and a single A. ostenfeldii cell, containing a Dinophysis sp. dinoflagellate cell. Several food vacuoles of ciliate origin had a red hue. This, together with the resemblance of A. ostenfeldii cells to planozygotes, suggests that similar structures previously identified as accumulation bodies may in fact be food vacuoles and that feeding may in some cases be associated with sexual processes.     

Intracoelomic parasitic Sporozoa in the burrowing spatangoid echinoidEchinocardium cordatum: coelomocyte reaction and formation of brown bodies

Echinocardium cordatum frequently harbours in its coelomic cavity the protozoan parasiteLithocystis schneideri. Motile stages of this gregarine (trophozoites and gamonts) may be surrounded by echinoid coelomocytes which show a peculiar transformation of their shape (i.e. each cell develops a single spine-like extension giving a pincushion aspect to the parasite). Encysted stages of the gregarine (gametocysts and sporocysts) are found mostly within brown bodies. Brown bodies are particular mesothelium-covered formations occurring usually in the coelomic cavity ofE. cordatum. It is suggested that brown bodies naturally originate from detached fragments of mesenteries.     

Flow cytometry studies of recombinant Escherichia coli in batch and continuous cultures: DNA and RNA contents; light-scatter parameters

Flow cytometry has been used to study the contents of macromolecular compounds and light-scatter parameters in batch and continuous cultures of a recombinant Escherichia coli strain that forms protein inclusion bodies. Changes in relative DNA and RNA contents and cell mass as estimated by forward-angle light scatter were detected and tightly correlated in batch culture. In addition, heterogeneity of wide-angle light scatter (WALS), which we related to the presence of cellular inclusion bodies, was observed. In contrast, the relative RNA content and cell mass did not change during continuous culture, and homogeneity of WALS was found. In addition, unexpected changes in relative DNA content were observed after 67 h of culture, indicating a change in bacterial physiology.     

Apoptosis of L929 Cells by Etoposide: A Quantitative and Kinetic Approach

Exponentially growing L929 cells were continuously exposed to 1 or 10 μMetoposide (VP-16). The effects of such treatment on cell growth, cycle distribution, morphology, and selected biochemical events were examined. DNA synthesis rates were markedly decreased and the protein/DNA ratio increased (unbalanced growth). Growth was blocked, with most cells being cycle arrested by 24 h in (late S–)G2–M. An asynchronous process of cell death then developed. Cells initially shrank into eosinophilic, trypan blue-excluding bodies, which were then released into the medium, and eventually became permeable to trypan blue. Transmission electron microscopy confirmed that dying cells acquired an apoptotic morphotype, with compaction and margination of chromatin, loss of microvilli, and shrinkage of cytoplasm and nucleus. Tissue transglutaminase activity and intensity of immunostaining rapidly increased in treated cultures. Internucleosomal DNA fragmentation could not be detected by agarose gel electrophoresis, yet flow cytometry revealed that the apoptotic bodies had a very low DNA fluorescence (≤10% of the 2nvalue). In agreement with the microscopic findings, this suggested that extensive DNA degradation had occurred in dead cells. While rates of cell loss from the monolayer amounted to 21 and 57% day⁻¹(1 and 10 μMVP-16, respectively), apoptotic indexes largely underestimated the extent of the process. These indexes only measured the accumulation of apoptotic bodies, i.e., the balance between their generation and disposal. The latter occurred by mechanisms similar to those that operate in tissues: “secondary necrosis” or phagocytosis by viable homotypic cells in the monolayer (“homophagy”).     

Cytotoxic effects of β‐asarone on Sf9 insect cells

β‐Asarone is the predominant component of the essential oil of rhizomes of Acorus calamus Linn ( Sweet flag). Although rhizome extracts from this plant have long been used for insect pest control, their cytotoxic effects on insect cells are not well understood. In this study, we evaluated the potency of β‐asarone as a natural insecticide by using a Spodoptera frugiperda cell line (Sf9). To assess the cytotoxic effects of β‐asarone on Sf9 cells, we observed morphologic changes in treated cells and performed a cell proliferation assay and a DNA fragmentation assay. After 24 and 48 h of treatment with β‐asarone, the proliferation of the Sf9 cells was inhibited in a dose‐dependent manner, with IC₅₀ values of 0.558 mg/ml at 24 h and 0.253 mg/ml at 48 h. Morphologic changes in β‐asarone‐treated cells were typical of apoptosis and included loss of adhesion, cell shrinkage, and small apoptotic bodies. The DNA laddering present in β‐asarone‐treated SF9 cells and annexin V assay confirmed that this compound can induce apoptosis in insect cells. Together, these findings suggest that apoptosis induction may be one mechanism through which β‐asarone inhibits the proliferation of insect cells and thus exerts insecticidal effects.     

Fusaric acid induces apoptosis in saffron root-tip cells: roles of caspase-like activity, cytochrome c, and H2O2

Programmed cell death (PCD), now known as apoptosis, is accompanied by specific morphological features. In this study, fusaric acid, a fusarium mycotoxin, was used to examine cell death in saffron (Crocus sativus Linnaeus) roots, using several apoptosis assays. Our results show that moderate FA doses (50–100 μM) induce apoptotic features while high FA doses (> 200 μM) stimulate necrosis. The apoptotic-like features induced by moderate doses of FA include chromatin condensation, formation of condensed chromatin spheres which bud from the nucleus, fragmentation of nucleosomal DNA into ∼ 180 bp fragments, exposure of phosphatidyl serine to the external membrane leaflet, delivery of cytochrome c to cytosol, and generation of H₂O₂. These apoptotic alterations in root cells are not observed in the presence of serine protease, caspase-1 or caspase-3 inhibitors. It is proposed that production of H₂O₂ and release of cytochrome c into the cytosol may activate caspase-like proteases and thus establish the apoptotic pathway. As nuclei budding spheres formed in plant root cells after exposure to 50–100 μM FA doses seem to be digested inside the cytosol, we suggest labeling them as internal apoptotic bodies (IAB) that may be more informative than previously used term, apoptotic-like bodies.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Isolation and partial characterization of conditional cell division cycle mutants inChlamydomonas

Summary We have isolated a number of temperature conditional cell division cycle mutants of the unicellular plantChlamydomonas reinhardtii that are defective in single nuclear genes. Cells grow and divide normally at the permissive temperature (21 °C), but arrest in division at the restrictive temperature (33 °C). We have characterized these mutants using DNA probes and immunofluorescence techniques to localize cytoskeletal and microtubule organizing centre proteins. We describe here 3 broad classes of cell cycle mutation which result in cell cycle arrest with: unreplicated DNA (G1 arrest), duplicated DNA (G2 arrest) and multiple nuclei due to defective cytokinesis (cytokinesis arrest). The continuation of nuclear division in mutants blocked in cytokinesis provides support of an earlier hypothesis that stage specific events in theChlamydomonas cell cycle are arranged in separate dependent sequences. The mutants isolated in the present study provide insights into the role of cytoskeletal proteins in the coordination of plant cell division and the means to investigate the molecular mechanisms whereby division by multiple fission is controlled in the unicellular plantChlamydomonas.Abbreviations BB basal bodies - EMS ethylmethane sulphonate - MT microtubule - MTOC Microtubule organizing centre - NBBC nucleus-basal body connector - PAR photosynthetically active radiation     

Ultrastructure and Development of Sclerotia of Botrytis cinerea Pers. in vitro

The development of sclerotia of Botrytis cinerea was examined at four stages during their maturation. The surface structure developed a network of profusely branched hyphae through their coalescence to a compact sclerotial body which was maturated by the deposition of melanin pigment. A characteristic feature of the hyphal cells of B. cinerea during the later stages of development was the presence of paramural bodies (plasmalemmasomes and lomasomes). Electrondense bodies with a limiting double-membrane congregated against the transverse septa of hyphal cells as sclerotia matured and may migrate from cell to cell through septal pores. We suggest that these and the lipid bodies found in hyphal cells may have a storage function in the resting sclerotia.     

NUCLEOCYTOPLASMIC RATIO REQUIREMENTS FOR THE INITIATION OF DNA REPLICATION AND FISSION IN TETRAHYMENA

Hydroxyurea (10 mM) arrests the exponential growth of Tetrahymena by blocking DNA replication during S-phase. After removal of the hydroxyurea (HU), they have a long recovery period during which they are active in DNA synthesis. ³H-TdR uptake showed that on completion of the recovery period, the cells divide (recovery division) and enter a cell cycle which lacks G₁. The frequency, size and DNA content of the extranuclear chromatin bodies (ECB) formed at this division are all markedly increased (2–4) over the corresponding values obtained from exponential growth phase controls. Microspectrophotometric analysis of macronuclear DNA content (N) coupled with the cytoplasmic dry mass (C) values suggest that specific N to C ratios (N/C) are required for the initiation of DNA replication and fission: during a normal (exponential growth) cell cycle, both N and C double, but asynchronously, so that the N/C of both post-fission-daughter cells and pre-fission cells is identical (standardized to N/C = 1) but late G₁ cells have a low N/C. During a 10 hr exposure to HU, the N remains essentially the same whereas the C increases. When the HU is removed, the N increases by 4× and the C continues to increase until just prior to recovery division when it also reaches a value 4× that of the original daughter cells. Thus, the N/C = 1 is re-established. The enlarged ECB formed during recovery division may function to lower the N/C in the daughter cells, which in turn may in some way stimulate immediate DNA replication, thus eliminating G₁. The elimination of G₁ (and shortening in a few subsequent cell cycles) allows less time for cytoplasmic growth and results in the return of the cells to the generation time and the N and C values observed prior to the HU treatment.     

The fine structure of blood cells in the ascidian Perophora viridis

The fine structure of each of the blood cell types of Perophora viridis has been characterized and strong evidence for localization of vanadium in two of these types is given. There are eight cell types; phagocytes which may contain completely engulfed cells, lymphocytes with a prominant nucleolus and scanty cytoplasm packed with clustered ribosomes, and six other cell types each with distinctive granules. Morula cells contain a central nucleus and cytoplasm filled by wedged bodies, about five of which are seen in section. These bodies contain regularly spaced electron dense foci. Green cells have the same organization but contain bodies which are electron dense throughout. Granular amoebocytes contain many smaller lightly staining oval bodies and much glycogen. Another cell type (probably orange cells of light microscopy) contains numerous granular rounded bodies. Compartment cells have vacuoles containing electron dense particles and signet ring cells have usually one large vacuole which is electron dense lined and may contain electron dense particles. Developmental stages of these cell types show involvement of endoplasmic reticulum and Golgi bodies in granule formation. After glutaraldehyde fixation alone the only extremely electron dense components are particles in the compartment cells and signet ring cells implicating these as sites of vanadium localization, although not excluding other cell types.