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Biomedical subjects

E Kellenberger

Publications and source records attributed to E Kellenberger.

At least 37 records · Page 2Linked to original sources
PubMed1992

Artefacts and morphological changes during chemical fixation.

The normally 'condensed' (darkly stained) chromosomes of dinoflagellates decondense by swelling. This occurs in an increasing number of cells when the concentration of added OsO4 is decreased. With different fixatives other types of disintegration can be observed, which vary with the concentration. With cryofixation and freeze-substitution the chromosomes are most 'condensed'. Escherichia coli infected with bacteriophage T4, with or without active lysozyme production, were studied by optical densitometry for partial lysis and by light and electron microscopy for observing swelling. When active lysozyme is present some of the acrolein (2.5%)-glutaraldehyde (2%)-fixed cells swell at 0 degrees C, but do not in the absence of lysozyme nor when fixed at room temperature. If OsO4 is added at concentrations < or = 0.5%, partial lysis occurs when lysozyme is present. The optical density decreases, the cells lose some matter and swell slightly. The corresponding electron micrographs show gap formation by curdling and/or a decreased concentration of the cytoplasm which reveals certain phage-related particles.

Animals↗
PubMed1991

Storage, ultrastructural targeting and function of toposomes and hyalin in sea urchin embryogenesis.

This study compares by immunogold labeling the ultrastructural localization of a hexameric 22S glycoprotein, called toposome, with that of hyalin in unfertilized eggs and cells of hatched sea urchin blastulae. Nearly all hyalin is present in the electron translucent compartment of the cortical granules and in the translucent non-cortical pigment granules. In the blastula both of these intracellular stores have vanished and hyalin now forms a broad band below the apical lamina. By contrast, in the egg toposomes are present on the surface, as well as stored in yolk granules and in the electron dense lamellar compartment of the cortical granules. In the hatched blastula, toposomes that have been modified by limited proteolysis in the yolk granules, are associated with the plasma membranes of all newly formed cells, while the toposomes originating from the cortical granules have been incorporated as unmodified 160 kDa polypeptides into an extracellular double layer enveloping the embryo on the outside of the hyaline layer. From evidence discussed in detail, we conclude that the extracellular toposomes rivet the apical lamina to the surface and underlying cytoskeleton of the microvilli, while the modified toposomes from the yolk granules are responsible for position specific intercellular adhesion as they are released to the surface of newly formed cells. We propose that all the material stored in yolk granules is utilized for the assembly of new membranes.

Animals↗
PubMed1991

Physiological, morphological, and physicochemical characterization of a novel Escherichia coli bacteriophage, phage MM.

A double-stranded DNA containing, T even-like, Escherichia coli bacteriophage, called MM, has been isolated from the local sewage and purified by polyethylene glycol precipitation followed by banding on a cesium chloride three-step gradient. It yields a burst size of 75 particles per infected cell, and has an adsorption coefficient of 3.3 x 10(-10) cm3/min and a latent period of 45 min. Electron microscopy of phage MM reveals an isometric icosahedral head, 92 nm long and 81 nm wide, and a 112-nm-long contractile tail with six pairs of 40-nm-long fibers attached to its baseplate. Phage MM appears similar to E. coli phage T4 or Salmonella phage O1. The density of phage MM in cesium chloride is 1.515 g/ml, and its total mass is 144 MDa. Gel electrophoresis of purified MM capsids displays two major capsid proteins in approximately equimolar amounts and with apparent molecular masses of 38 and 15 kDa. Similarly, purified MM tails yield two major polypeptides with apparent molecular masses of 55 and 16 kDa, most likely representing the major tail sheath and tail tube polypeptides. Its double-stranded DNA has a G-C content of 50%, a length of 131 kilobases (kb), and a mass of 89 MDa.

Adsorption↗
PubMed1991

Mass analysis of bacteriophage T4 proheads and mature heads by scanning transmission electron microscopy and hydrodynamic measurements.

Quantitative mass analysis of bacteriophage T4 proheads by scanning transmission electron microscopy (STEM) revealed a mass of 79.5 +/- 0.6 MDa, while hydrodynamic measurements yielded a prohead mass of about 80 MDa. This is 25% less than the prohead mass deduced from its polypeptide composition, and this finding implies that the bacteriophage T4 prohead is built of fewer polypeptide copies than previously reported. In contrast, the mass of mature heads measured by STEM, 194 +/- 2 MDa, is in agreement with previous mass measurements of DNA and protein content, and it is consistent with the previously determined stoichiometry. This good agreement of average STEM values for proheads and mature heads with corresponding hydrodynamic measurements suggests that STEM allows faithful evaluation of the masses of large supramolecular assemblies (i.e., greater than or equal to 200 MDa) such as whole viruses or cellular organelles.

Formaldehyde↗
PubMed1991

The potential of cryofixation and freeze substitution: observations and theoretical considerations.

The theoretical and experimental evidence in favour of cryofixation and freeze-substitution are critically reviewed. The solubility of macromolecules in water is due to the hydration shells. Their behaviour at different temperatures and the consequences of their removal during the processing for embedding are explained. Gelation prior to the transfer into solvents prevents macromolecules aggregating. During substitution at low temperatures, DNA is gelled, justifying the use of the term cryofixation. It is proposed that the preservation of hydration shells at the lowest temperature, and their transformation into minute gaps after a rise of temperature, facilitates the exhibition of epitopes.

Animals↗
PubMed1991

Coralline shape of the bacterial nucleoid after cryofixation.

A new procedure of immunostaining sections of cryofixed and freeze-substituted Escherichia coli shows that DNA extends from its bulk into small ribosome-free spaces throughout the cytoplasm, resulting in a coralline-shaped nucleoid. Low-resolution imaging of a bacterium reconstructed from serial sections demonstrated that the small excrescencies are not resolved. The resulting photograph shows the same features as phase-contrast light micrographs.

Chromatin↗
PubMed1990

Form determination of the heads of bacteriophages.

The shape of the DNA-containing heads of many bacteriophages is not only determined by the properties of the protein subunits which build the shell (capsid) but also by the scaffolding core which is a transient structure of the prohead. The form-determining properties of the scaffolding proteins have been characterized by genetic methods based on conditional mutants and site-directed mutagenesis. The mechanism of form determination has been studied by in vitro assembly experiments. The theoretical background is discussed and different models for mechanisms of form determination are considered. Definitive decisions about the validity of a model is still limited by the difficulty of obtaining unambiguous answers on the stoichiometry and the fine structure of the scaffold because of their high instability.

Bacteriophages↗
PubMed1990

The 'Bayer bridges' confronted with results from improved electron microscopy methods.

In electron micrographs of conventionally prepared thin sections of Escherichia coli one observes (i) a wavy appearance of the two membranes showing frequent appositions (named adhesion sites) and (ii) intermembrane bridges after plasmolysis which, it is claimed, occur at the adhesion sites and are related to intermembrane protein transport (transmigration). When chemical fixation is replaced by cryofixation, the observations are very different. (a) The two membranes are equally spaced and no contacts, adhesions or other sorts of connections are visible. (b) After plasmolysis the protoplast is shrunken, but the typical bridges are no longer produced. (c) In addition, when peptidoglycan is stained on conventionally prepared sections, it is revealed as a 7-nm-thick sacculus which is not interrupted at the sites of apposition. In view of the new observations, the structural concepts derived from conventionally prepared material must be revised. It is proposed that the intermembrane space is entirely filled by a gel, the outer part of which is the 7 nm thick, very stable, chemically resistant peptidoglycan (or murein). The inner part is much less stable and is proposed to undergo rapid autolytic changes upon cell death. The large 'Bayer bridges' might then tentatively be explained as an artificial post-mortem enhancement of either a stream of proteins transmigrating across the periplasm or of a pre-existing, but not yet resolved, structure. This enhancement probably occurs during the 7-10 min between plasmolysis and fixation that are prescribed for the procedure necessary for revealing 'Bayer bridges'.

Bacterial Adhesion↗
PubMed1988

About the organisation of condensed and decondensed non-eukaryotic DNA and the concept of vegetative DNA (a critical review).

Experiments are reviewed that allow one to assign naturally occurring DNA-containing plasmas to either of two classes by virtue of their sensitivity to aggregation upon dehydration in organic solvents. The interphase nuclei of higher cells are relatively insensitive, while the DNA plasmas represented by bacterial nucleoids, vegetative bacteriophage and the chromosomes of dinoflagellates are sensitive. In higher cells the bulk of DNA is organised with histones in the form of nucleosomes. In prokaryotes and in the pool of vegetative phage DNA the most abundant histone-like protein HU is not associated with the bulk DNA, but localised in the border region with ribosomes where transcription and translation occur. These experimental results strongly suggest that the two classes of DNA plasmas are distinguishable by a low (1:10) or high (1:1) protein-to-DNA ratio. The hypothesis is formulated that the vegetative DNA (replicating and transcribing), throughout the living world, is nucleosome-free; during evolution, nucleosomes would have been introduced as a simple and adequate means for compacting the resting DNA. Condensation of DNA does not occur with prokaryotic nucleoids, but does take place when DNA is withdrawn from the vegetative phage pool to become packaged into phage heads. Dinoflagellate chromosomes are rather condensed although structurally different from eukaryotic chromosomes (e.g., those from Euglena) and are much more aggregation-sensitive.

Bacteria↗
PubMed1988

Head structure of bacteriophages T2 and T4.

The length-to-width ratios of bacteriophage T2 and T4 heads and stereometric angles specifying the prolate icosahedral T2 capsid were evaluated on electron micrographs recorded from samples prepared by a variety of methods. The copy numbers of the major capsid protein, gp23*, of T2 and T4 phages were compared by quantitative gel electrophoresis. Taken together, the resulting values are most compatible with triangulation numbers T = 13 and Q = 21 for both T2 and T4, thus confirming the previously proposed capsid architecture of T4 revealed by indirect measurements and thereby eliminating the repeatedly reported discrepancy between T2 and T4 in favor of a common Q number of 21 corresponding to 960 copies of gp23*.

Capsid↗
PubMed1988

Intracellular location of the histonelike protein HU in Escherichia coli.

Immunocytochemical labeling of thin sections of cryosubstituted, Lowicryl-embedded Escherichia coli cells with protein A-colloidal gold was used to study the structural organization of the bacterial nucleoid. We found that the histonelike protein HU was not associated with the bulk DNA in the nucleoid but was located in areas of the cell where metabolically active DNA is associated with ribosomes and where single-stranded DNA, RNA polymerase, and DNA topoisomerase I were also located. The resolution of the methods used did not allow us to decide whether HU was associated either with ribosomes or with transcriptionally active DNA, nor could we demonstrate interaction of HU with either.

Bacterial Proteins↗
PubMed1988

Length and shape variants of the bacteriophage T4 head: mutations in the scaffolding core genes 68 and 22.

The shape and size of the bacteriophage T4 head are dependent on genes that determine the scaffolding core and the shell of the prohead. Mutants of the shell proteins affect mainly the head length. Two recently identified genes (genes 67 and 68) and one already known gene (gene 22), whose products are scaffold constituents, have been investigated. Different types of mutants were shown to strongly influence the proportion of aberrantly shaped particles. By model building, these shape variants could be represented as polyhedral bodies derived from icosahedra, through outgrowths along different polyhedral axes. The normal, prolate particle is obtained by elongation along a fivefold axis. The mutations of the three core genes (genes 67, 68, and 22) affect the width mainly by lateral outgrowths of the prolate particle, although small and large isometric particles are also found. Many of the aberrant particles are multitailed, suggesting a correlation between tail attachment sites and shape.

DNA Mutational Analysis↗
PubMed1987

Isolation and reassembly of bacteriophage T4 core proteins.

The products of genes 22, 67 and 68, and the internal proteins IPI, IPII and IPIII, as components of the scaffolding core of the bacteriophage T4 prohead, have been isolated and purified by hydroxylapatite column chromatography. Under conditions promoting reassembly in vitro, the proteins associated into elongated particles of practically constant width but variable length that we have called polycores. Preliminary optical diffraction experiments indicate that polycores may have an ordered structure, possibly helical, as has been suggested for the polyhead core. The coassembly of core proteins and the purified shell protein gp23 results in the formation of core-containing polyheads. Occasionally, prolate core-like particles have been observed but their reproducible formation has not been attained. Attempts to investigate the role of the minor prohead component gp20 in core assembly have been made through the cloning of the corresponding gene in an expression vector and subsequent purification of the protein.

Chromatography↗
PubMed1987

Organization of double-stranded DNA in bacteriophages: a study by cryo-electron microscopy of vitrified samples.

In this paper it is shown that conformation and packing of double-stranded DNA within the head of bacteriophages lambda and T4 can be assessed by cryo-electron microscopy of vitrified specimens. Electron diffraction patterns show that DNA within vitrified bacteriophages has a B conformation. Electron micrographs of vitrified bacteriophages show domains within the head formed by a approximately 2.5-nm striation and arising from the DNA packing. The number of differently oriented domains seen within a vitrified bacteriophage depends upon the geometry of the DNA container: the bacteriophage capsid. The packing of DNA within bacteriophages seems then to be governed by at least two phenomena. The first is the tendency of DNA to form local alignments (nematic liquid crystals). The second is the orientation of these liquid crystals by the bacteriophage capsid. From these observations we propose a possible packaging mechanism: constrained nematic crystallization.

Bacteriophage lambda↗
PubMed1987

Use of on-section immunolabeling and cryosubstitution for studies of bacterial DNA distribution.

Escherichia coli cells were very rapidly frozen and substituted at a low temperature with 3% glutaraldehyde in acetone. Infiltration and embedding with Lowicryl K4M were carried out at -35 degrees C. This procedure resulted in good structural preservation of both the nucleoid morphology and its DNA plasm, such that immunolabeling with the protein-A gold technique could be carried out. With antibodies specific for either double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA), it was shown that dsDNA was present throughout the nucleoid but that ssDNA was located on the nucleoid periphery. Chloramphenicol-treated cells, in which protein synthesis but not DNA replication is stopped, produced a characteristic ringlike nucleoid shape and had both dsDNA and ssDNA present throughout the annular section of the DNA plasm. The relationship between metabolically active DNA and overall bacterial genome organization is discussed.

Antibodies, Monoclonal↗
PubMed1987

The efficiency of immunolabel on Lowicryl sections compared to theoretical predictions.

The surface of thin sections of aldehyde-fixed biological material shows a specimen-related relief of 2-6 nm with Lowicryl. Epon sections are about three times smoother. The relief is the consequence of thin-sectioning being in reality a cleavage. Epitopes are supposed to be laid open (or set free) because cleavage follows the interfaces between protein and Lowicryl. We have developed a simple theory on this basis and have theoretically estimated the efficiency of on-section labeling and compared it with experimental data. For randomly dispersed proteins in cytoplasm, Lowicryl sections will yield significant label only when the concentration of the antigen is about 10 microM or more. The complex situation of more compact proteins, as represented by fibers, sheets, and biological membranes is discussed and the difficulty of significant calculations is explained. Pre-embedding labeling and melted cryosections should give 10-30 times more label. The possible reasons for the observed much smaller gain of not more than two to three times are discussed.

Acrylic Resins↗