Loading a ring: structure of the Bacillus subtilis DnaB protein, a co-loader of the replicative helicase

Loading of the ring-shaped replicative helicase is a critical step in the initiation of DNA replication. Bacillus subtilis has adopted a two-protein strategy to load its hexameric replicative helicase: DnaB and DnaI interact with the helicase and mediate its delivery onto DNA. We present here the 3D electron microscopy structure of the DnaB protein, along with a detailed analysis of both its oligomeric state and its domain organization. DnaB is organized as an asymmetric tetramer that is comprised of two stacked components, one arranged as a closed collar and the other as an open sigma shape. Intriguingly, the 3D map of DnaB exhibits an overall architecture similar to the structure of the Escherichia coli gamma-complex, the loader of the ring-shaped processivity factor. We propose a model whereby each DnaB monomer participates in both stacked components of the tetramer and displays a different overall shape. This asymmetric quaternary organization could be a general feature of ring loaders.     

Clarifying the Taxonomic Identity of a Phylogenetically Important Group of Eukaryotes: Planomonas is a Junior Synonym of Ancyromonas

ABSTRACT. Ancyromonas was first described in 1882 by Saville Kent, with the modern concept of the genus dating from 1979 with the work of Hänel. Since then, organisms assigned to Ancyromonas have been found to be common in diverse ecosystems, and the group's isolated phylogenetic placement renders it of considerable evolutionary interest. However, in 2008 Cavalier‐Smith et al. concluded that all modern accounts of Ancyromonas were of a different organism from that described by Saville Kent, and erected the new genus Planomonas to encompass modern observations of Ancyromonas, and several new species. We critique the rationale for creating this new genus, reexamining the original sources and making additional observations using light and electron microscopy. We find that almost all the differences between the genera are mistaken or insubstantial. In particular, (1) Cavalier‐Smith et al. characterized Ancyromonas sensu Saville Kent as anchoring and Planomonas as gliding, while we find that each type of organism actually does both, and (2) it was claimed that Planomonas is flattened while Ancyromonas sensu Saville Kent is not, but this argument is inconsistent. We treat Planomonas as a junior synonym of Ancyromonas, and Planomonas mylnikovi as a junior synonym of Ancyromonas sigmoides. We transfer Planomonas cephalopora, Planomonas micra, Planomonas howeae and Planomonas limna to Ancyromonas. The genus Ancyromonas therefore includes: A. sigmoides, Ancyromonas cephalopora n. comb., Ancyromonas melba, Ancyromonas sinistra, Ancyromonas micra n. comb., Ancyromonas howeae n. comb., and Ancyromonas limna n. comb.     

Ultrastructure and cytochemistry of the gastric mucosa of a reptile,Tiliqua scincoides

Summary The gastric mucosa of a reptile, the lizard Tiliqua scincoides, has been examined by light and electron microscopy. The gastric pits lead into glands that are extensively coiled in the proximal stomach but become progressively shorter and straighter in the distal stomach. The following epithelial cell types have been identified: (i) Surface mucous cells (SMC) line the entire lumenal surface as well as the pits. They contain mucus granules that stain with periodic acid-Schiff and, like the granules of mammalian SMC, commonly contain an electron dense core that appears not to be mucus (periodic acid-chromic acid-silver methenamine nonreactive). (ii) Glandular mucous cells are present in glands throughout the mucosa. They are probably homologous with the mucous neck and antral gland cells of mammals; like SMC their mucus granules contain nonglycoprotein cores. (iii) Oxynticopeptic cells (OPC) are the predominant cell type in the proximal glands but become infrequent distally. Their fine structure resembles that of OPC in other nonmammalian vertebrates, with features like those of both parietal cells and zymogen cells of mammals, (iv) Endocrine cells of three different types have been identified. Two of these show close similarities to the EC and ECL cells of mammals.The authors thank Mrs. D. Flavell for technical assistance. This study was supported by a grant from the Clive and Vera Ramaciotti Foundations     

How a long linear molecule can be straightened on a support

A relatively simple means is proposed for depositing a macromolecule such as DNA on a dielectric support in a straight line (which is essential for “fast reading” of sequences by physical methods). A randomly folded molecule in solution can be caused to align with a “straightening electrode” under the support, as in a phase transition. Conditions are envisaged that should ensure processive settling without defects (loops/knots).     

Development of a convenient and supersensitive high-throughput screening system for genetically encoded fluorescent probes of small molecules using a confocal microscope

Monitoring the dynamic patterns of intracellular signaling molecules, such as inositol 1,4,5-trisphosphate (IP₃) and Ca²⁺, that control many diverse cellular processes, provides us significant information to understand the regulatory mechanism of cellular functions. For searching more sensitive and higher dynamic range probes for signaling molecules, convenient and supersensitive high throughput screening systems are required. Here we show the optimal “in Escherichia coli (E. coli) colony” screening method based on the twin-arginine translocase (Tat) pathway and introduce a novel application of a confocal microscope as a supersensitive detection system to measure changes in the fluorescence intensity of fluorescent probes in E. coli grown on an agar plate. To verify the performance of the novel detection system, we compared the changes detected in the fluorescent intensity of genetically encoded Ca²⁺ indicator after Ca²⁺ exposure to two kinds of conventional fluorescence detection systems (luminescent image analyzer and fluorescence stereomicroscope). The rate of fluorescence change between Ca²⁺ binding and unbinding detected by novel supersensitive detection system was almost double than those measured by conventional detection systems. We also confirmed that the Tat pathway-based screening method is applicable to the development of genetically encoded probes for IP₃. Our convenient and supersensitive screening system improves the speed of developing florescent probes for small molecules.     

Unraveling the organization of the internal nuclear matrix: RNA-dependent anchoring of NuMA to a lamin scaffold

Using quantitative immunoelectron microscopy we show here that when the nuclear matrix is isolated from rat hepatocytes in the presence of an inhibitor of RNase activity both lamins and the nuclear mitotic apparatus protein (NuMA) preferentially localize within the electron-dense domains of the internal nuclear matrix (INM). After RNA digestion NuMA undergoes a sharp depletion, while labeling by an antibody against lamins A and C within the electron-transparent regions increases, suggesting that a subset of lamin epitopes is masked by the interaction with RNA. We were able to explain this result by visualizing for the first time a thin web of lamin protofibrils which connects the electron-dense regions. Confirmation of these changes has been obtained by immunoblot analysis and confocal microscopy. As RNA digestion results both in the release of NuMA and in the collapse of the INM, we propose that a fraction of nuclear RNA brings about the association of NuMA islands with a lamin scaffold and that this interaction is required to maintain the latter in a state of high molecular dispersion.     

Polarization-resolved SHG imaging as a fast screening method for collagen alterations during aging: Comparison with light and electron microscopy

Our previous study on rat skin showed that cumulative oxidative pressure induces profound structural and ultrastructural alterations in both rat skin epidermis and dermis during aging. Here, we aimed to investigate the biophotonic properties of collagen as a main dermal component in the function of chronological aging. We used second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) on 5 μm thick skin paraffin sections from 15-day-, 1-month- and 21-month-old rats, respectively, to analyze collagen alterations, in comparison to conventional light and electron microscopy methods. Obtained results show that polarization-resolved SHG (PSHG) images can detect collagen fiber alterations in line with chronological aging and that this method is consistent with light and electron microscopy. Moreover, the β coefficient calculated from PSHG images points out that delicate alterations lead to a more ordered structure of collagen molecules due to oxidative damage. The results of this study also open the possibility of successfully applying this fast and label-free method to previously fixed samples.     

Isolated generative cells in some angiosperms: a further study

Summary Generative cells were isolated from the pollen grains of three angiosperm species by a method similar to that previously reported for Haemanthus katherinae (Baker). Both the external appearance and the internal structure of the isolated generative cells were observed by light and scanning electron microscopy. The dynamic changes occurring in the cells after they had been liberated from the pollen grains were recorded by video-enhanced microscopy. The distribution of microtubules in the isolated cells was revealed by immunofluorescence.This work was done during the senior author's sabbatical leave at the Department of Biological Sciences, Dartmouth College, Hanover, N.H. (USA) and this paper is dedicated to Professor R.D. Allen, who did not live to see this work completed but who was directly involved in supporting this project.     

Discotricha papillifera: Structure and Morphogenesis of a Marine Interstitial Ciliate*

SYNOPSIS. Discotricha papillifera Tuffrau, a marine interstitial ciliate, is redescribed with the aid of light, scanning, and transmission electron microscopy from cells collected at a New Hampshire beach. Presence of primitive membranelles as well an an advanced stomatogenesis is demonstrated. The ultrastructure, including a unique membrane-bounded septate structure, is described. The cell is tentatively placed in the nassulid suborder Microthoracina, but affinities with other groups are discussed.     

Morphological features of osteoclasts derived from a co-culture system

The interaction between the receptor activator of NfKB (RANK) and its ligand receptor activator of NfKB ligand (RANKL) has recently been proven to be pivotal for osteoclast differentiation and activation. The influence of RANK-RANKL signaling on osteoclast formation was established by co-culturing murine osteoblasts (type CRL-12257) and murine mononuclear monocytes (RAW 264.7). The aim of the present study was to examine, by means of morphological techniques, the interaction between these two cell lines grown in the absolute absence of exogenous cytokines and other stimulating factors. Moreover, we wanted to show that our model could provide a system to analyze the bone resorption process. Mineralized matrix induced morphological changes of osteoclasts (OC) by the formation of organized ruffled-border and a large number of secondary lysosomal vesicles. On the contrary, OC grown on glass coverslips without dentin showed no organized ruffled border or secondary lysosomes. The study of the relationship between these two cell types could establish new approaches for a potential pharmacological control of these cell types and tissues in health and disease.     

Nuclear localization of nucleocapsid-like particles and HCV core protein in hepatocytes of a chronically HCV-infected patient

Little is known about the life cycle of hepatitis C virus. Determination of the subcellular localization of HCV proteins may contribute to our understanding of the in vivo functions of the viral proteins. HCV core protein regulates multiple functions in host cells and it has been detected both in the cytoplasm and in the nucleus using different expression systems. In this study, nucleocapsid-like particles were observed in the nucleus of hepatocytes from a chronically HCV-infected patient. They were similar in size and shape to those of HCV core-like particles purified from recombinant Pichia pastoris cells. In addition the HCV core protein was detected not only in the cytoplasm but also in the nucleus and nucleolus of hepatocytes by immunoelectron microscopy. This is the first report showing nuclear localization of HCV core protein and nucleocapsid-like particles in hepatocytes during in vivo HCV infection.     

Light and electron microscope study of a new species of Thelohania (Microsporida) in the shrimp Pandalus jordani

Thelohania butleri n. sp. was found in cells of skeletal muscles of the shrimp Pandalus jordani, from Queen Charlotte Sound, British Columbia, Canada. Sporulation stages were studied with the light and the electron microscope. Earliest stages were small and apparently uninucleate. Next were small diplokaryotic cells that possibly arose by fusion of the former. These enlarged and underwent sporogony. Sporogony was a series of three binary divisions, each producing unikaryotic cells. There was no sporogonial plasmodium. The spore was ovoid, 4.8 × 3.1 μm (stained), with a large crescentic nucleus and rounded posterior vacuole. The polar filament was isofilar, doubly coiled, with about 10 turns. This species closely resembles the type T. giardi Henneguy. It is concluded that sporogony by means of three binary divisions and lack of a sporogonial plasmodium may be essential characters of the genus Thelohania Henneguy, 1982.     

Modification of membranes by quercetin, a naturally occurring flavonoid, via its incorporation in the polar head group

Quercetin is a naturally occurring flavonoid that has a lot of beneficial properties to human health. In this report, using the spin label technique, the influence of quercetin on the fluidity of multilamellar DPPC liposomes was studied. The polarity of the environment preferred by quercetin was also examined by determining the dependence of the position of electronic absorption maxima on dielectric properties of different environments. Autofluorescence of quercetin was also used to examine its distribution in cells. An additional aim of the study was to find how quercetin presence affects human skin fibroblasts. The results showed that incorporation of quercetin at physiological pH into DPPC liposomes caused changes in the partition coefficient of the Tempo spin label between water and polar head group phases. By determining the electronic absorption maxima, we observed that the chromophore of quercetin is localized in the polar head region. Fluorescence microscopy of HSF cells showed quercetin presence in the membrane, cytoplasm and inside the nucleus. Ultrastructural observation revealed some changes, especially in membranous structures, after flavonol treatment. From the results we have concluded that quercetin present in the membrane and other structures can cause changes within cells crucial for its pharmacological activity.     

Label-free detection and dynamic monitoring of drug-induced intracellular vesicle formation enabled using a 2-dimensional matched filter

Analysis of vesicle formation and degradation is a central issue in autophagy research and microscopy imaging is revolutionizing the study of such dynamic events inside living cells. A limiting factor is the need for labeling techniques that are labor intensive, expensive, and not always completely reliable. To enable label-free analyses we introduced a generic computational algorithm, the label-free vesicle detector (LFVD), which relies on a matched filter designed to identify circular vesicles within cells using only phase-contrast microscopy images. First, the usefulness of the LFVD is illustrated by presenting successful detections of autophagy modulating drugs found by analyzing the human colorectal carcinoma cell line HCT116 exposed to each substance among 1266 pharmacologically active compounds. Some top hits were characterized with respect to their activity as autophagy modulators using independent in vitro labeling of acidic organelles, detection of LC3-II protein, and analysis of the autophagic flux. Selected detection results for 2 additional cell lines (DLD1 and RKO) demonstrate the generality of the method. In a second experiment, label-free monitoring of dose-dependent vesicle formation kinetics is demonstrated by recorded detection of vesicles over time at different drug concentrations. In conclusion, label-free detection and dynamic monitoring of vesicle formation during autophagy is enabled using the LFVD approach introduced.     

Rapid and sensitive detection method of a bacterium by using a GFP reporter phage

A rapid, sensitive, and convenient method for detecting a specific bacterium was developed by using a GFP phage. Here we describe a model system that utilizes the temperate Escherichia coli-restricted bacteriophage lambda, which was genetically modified to express a reporter gene for GFP to identify the colon bacillus E. coli in the specimen. E. coli infected with GFP phage was detected by GFP fluorescence after 4-6 hr of incubation. The results show that a few bacteria in a specimen can be detected under fluorescence microscopy equipped with a sensitive cooled CCD camera. When E. coli and Mycobacterium smegmatis were mixed in a solution containing GFP phage, only E. coli was infected, indicating the specificity of this method. The method has the following advantages: 1) Bacteria from biological samples need not be purified unless they contain fluorescent impurities; 2) The infection of GFP phage to bacteria is specific; 3) The fluorescence of GFP within infected bacteria enables highly sensitive detection; 4) Exogenous substrates and cofactors are not required for fluorescence. Therefore this method is suitable for any phage-bacterium system when bacteria-specific phages are available.     

Characterisation of a new strain of tobacco necrosis virus isolated from nutrient feeding solution*

A virus, isolated from the nutrient feeding solution of a bell pepper culture in a glasshouse, was characterised as a new serotype of tobacco necrosis necrovirus (TNV) and named TNV-nft. The virus was monopartite and contained a single RNA species and a single coat protein with molecular weights of 1.45± 10⁶ and 2.9 ± 10⁴ daltons, respectively. The particle had an apparent sedimentation coefficient of 110 S and a density of 1.37 g/cm³ in cesium chloride. In decoration tests antiserum prepared against TNV-nft reacted most strongly with the TNV strains Kassanis A and B, but at lower dilutions it also reacted with all other TNV-serotypes available. In Ouchterlony tests spur formation occurred with TNV strains Kassanis A and B, indicating that the TNV-nft strain was serologically distinct. The isolate was not associated with a satellite virus. Cloned cDNA prepared against viral RNA reacted exclusively with the viral RNA band in Northern blots.     

Device for holding a variety of tissue culture vessels during microscopy

Summary A device for holding tissue culture tubes, plates and flasks has been designed for use in microscopy of cell cultures, which eliminates changing holders when using varied types or sizes of vessel.