Electron microscopy of ageing cells of Pseudomonas rhodos: fine structure of native and isolated tubular membranes (author's transl)]

During a 10 day-incubation on agar surfaces at 30 degrees C, cells of the gram-negative soil bacterium Pseudomonas rhodos pass through three phases distinguishable by physiological and morphological criteria. When viewed by electron microscopy, typically "rolled" mesosomes could frequently be observed in young cells. In aged cells instead, loosely rolled or stretched-out, flattened tubules could be discerned, presumed to be degenerate mesosomes. Tubular flattened structures have been isolated from these cells by lysozyme treatment or sonication and were concentrated by differential centrifugation. Electron micrographs of these preparations showed long, straight tubules which sometimes appeared sealed at one end. Their width was 34 +/- 5 nm. They contained a lining of material, which could be digested by trypsin leaving behind an electron-transparent matrix. In rare cases, isolated tubules showed a periodic fine structure composed of ellipsoidal subunits. Optical diffraction analysis yielded a lattice consisting of subunits arranged in helices of pitch-angle 27 degrees; the unit cell dimensions were shown to be 112 X 56 A. Owing to their sensitivity to trypsin, components of the regular lattice are supposed to consist of protein. It is postulated that these protein components are layered onto a tubular membrane. These tubules are clearly distinguishable by their shape and fine structure from the periodic structure of a P. rhodos cell wall layer, which exhibits a tetragonal pattern, and also from polyheads and polysheaths of defective bacteriophages. Their possible origin from intact mesosomes in discussed.     

Nerve-muscle networks in the accessory gland tubules of a male insect: Structural and physiological properties

The accessory gland tubules of the cockroach Leucophaea maderae, (F.) are innervated by the phallic nerves which arise from the terminal ganglion. Tubule preparations stained with methylene blue showed that a fine network of nerve fibres covers the entire surface of the tubule with putative nerve cells at various points. Muscle fibres of the tubules were arranged in an irregular lattice and appeared as flattened elipses approx 8–10 μm across and 1.5 μm in thickness. These muscles were monomyofibrillar with a poorly developed T-tubule system and a diffuse Z band. Innervation of the muscles showed abundant evidence of neurosecretomotor-type terminals. Clear evidence was also obtained for a multiterminal-type innervation. Accessory gland tubules had a basic wave-like pattern of motion in vitro which showed a wide variation in the time-course of each swing (1–50 s). A twisting and curling motion of the tubules was also recorded. Spontaneous junctional potentials were observed in accessory gland preparations isolated from the terminal ganglion. The duration for junction potential ranged between 30–140 ms. Action potentials recorded from muscles had a slower rise time and a longer duration (230–530 ms).     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : II. Structure and Formation of Rhapidosomes

When cells of C. columnaris were broken open, treated with PTA, and examined in the electron microscope, tubular structures (rhapidosomes) were present in the preparations. The rhapidosomes are approximately 300 A in diameter. Their length varies from about 500 to about 15,000 A. An axial hole which runs the length of the rhapidosomes appears to widen and narrow with a regular periodicity. End-on views of short segments of rhapidosomes revealed the presence of subunits around their outside peripheries. The results of studies of lysed cells and of sectioned cells indicate that the rhapidosomes are produced during the disintegration of cells. It seems likely that the compound membranes of the mesosomes break down to give rise to the tubular structures. The mesosomal origin of rhapidosomes is postulated only for the rhapidosomes of C. columnaris, since the origin of rhapidosomes from other organisms was not investigated during this study. The rhapidosomes of C. columnaris may be unrelated to those of S. grandis, S. myxococcoides, A. violaceum, and Sorangium 495, since there was a difference in the details of fine structure between rhapidosomes from C. columnaris and those found in the other four organisms.     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : I. Structure and Formation of Mesosomes

Cells of Chondrococcus columnaris were sectioned and examined in the electron microscope after fixation by two different methods. After fixation with osmium tetroxide alone, the surface layers of the cells consisted of a plasma membrane, a dense layer (mucopeptide layer), and an outer unit membrane. The outer membrane appeared distorted and was widely separated from the rest of the cell. The intracytoplasmic membranes (mesosomes) appeared as convoluted tubules packaged up within the cytoplasm by a unit membrane. The unit membrane surrounding the tubules was continuous with the plasma membrane. When the cells were fixed with glutaraldehyde prior to fixation with osmium tetroxide, the outer membrane was not distorted and separated from the rest of the cell, structural elements (peripheral fibrils) were seen situated between the outer membrane and dense layer, and the mesosomes appeared as highly organized structures produced by the invagination and proliferation of the plasma membrane. The mesosomes were made up of a series of compound membranes bounded by unit membranes. The compound membranes were formed by the union of two unit membranes along their cytoplasmic surfaces.     

Effect of chromosomal breaks induced by x-irradiation on the number of mesosomes and the cytoplasmic organization of Streptococcus faecalis

A model which explains mesosome formation via a contraction of the cytoplasm and nucleoid when bacteria are physiologically disturbed was tested by: (1) X-irradiation of unfixed cells of Streptococcus faecalis to produce chromosomal breaks and to remove DNA attached to the cell membrane; (2) subsequent determination of the number of irradiated cells in which mesosomes (using electron microscopy) and central density changes (using phase-contrast microscopy) could be visualized after fixative was added. Results obtained by exposure of cells to doses up to 1100 krads before fixation indicated that: (1) the number of cells with central mesosomes was reduced proportional to the decrease in the molecular weight of the DNA due to double-strand breaks: (2) the number of cells with total (central plus peripheral) mesosomes and the number of cells with peripheral mesosomes were both reduced proportional to the removal of DNA attached to the cell membrane (M band); (3) the nucleoid became more diffusely organized. Exposure of cells to doses greater than 1100 krads before fixation resulted in: (1) an increase in the number of cells with central and peripheral mesosomes (compared to cells exposed to lower dosages); (2) a return to the centralized, dense nucleoid seen in unirradiated cells.These results suggest that mesosomes are formed when localized sites on the cell membrane are pulled from close contact with the cell wall into the cytoplasm by the action of a cross-linking fixative via the aggregation of intracytoplasmic components such as DNA. This model considers the attachment of DNA and/or other cytoplasmic components to the membrane as an intrinsic part of its mechanism. The formation of central and peripheral mesosomes in unirradiated and X-irradiated cells are contrasted.     

Regulation of chitin synthase activity inSaccharomyces cerevisiae: Effect of the inhibition of cell division and of synthesis of RNA and protein

The effect of pronase and trypsin on the activation or deactivation (degradation?) of chitin synthase ofSaccharomyces cerevisiae occurs faster in membranous preparations than in toluene-treated cells. When the temperature is raised, the former preparation is deactivated earlier than the latter one. The activity found in growing cells is not modified after inhibition of protein synthesis by cycloheximide or amino acid starvation or by the inhibition of RNA synthesis. It was possible to activate the chitin synthase ofS. cerevisiae cdc 25 grown at 23°C by means of pronase, whereas trypsin had no effect. After the cells were grown at 37°C, chitin synthase could not be activated either with trypsin or with pronase. This effect occurred independently of protein synthesis but did not take place when the cells were toluenized prior to the transfer at 37°C. These results suggest that the low catalytic levels and stability of the chitin synthase found in actively growing cells ofS. cerevisiae may be due to the restrictions introduced in the system by its membrane location.     

Über Geißelfeinstrukturen und Fimbrien bei zwei Pseudomonas-Stämmen

Summary Electron microscopical investigations of the flagella of Pseudomonas rhodos reveal a fine structure consisting of a left handed double helix.In Pseudomonas echinoides cell and flagellum are joined by a pinlike connecting element. Opposite to the flagellum a cluster of fimbriae is polarly inserted in cells of this strain. In stars the cells are held together by the fimbriae.     

Structure of Lipid Tubules Formed from a Polymerizable Lecithin

We have studied tubules formed from a polymerizable lipid in aqueous dispersion using freeze-fracture replication and transmission electron microscopy. The polymerizable diacetylenic lecithin 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine converts from liposomes to hollow cylinders, which we call tubules, on cooling through its chain melting phase transition temperature. These tubules differ substantially from cochleate cylinders formed by phosphatidylserines on binding of calcium. The tubules have diameters that range from 0.3 to 1 μm and lengths of up to hundreds of micrometers depending on conditions of formation. The thickness of the walls varies from as few as two bilayers to tens of bilayers in some longer tubules. Their surfaces may be either smooth, gently rippled, or with spiral steps depending on sample preparation conditions, including whether the lipids have been polymerized. The spiral steps may reflect the growth of the tubules by rolling up of flattened liposomes.     

Nuclear and cell division in Bacillus subtilis: cell development from spore germination.

The changes in the morphology of the nucleoids and the mesosomes in Bacillus subtilis cells during synchronous outgrowth after spore germination were followed in large-scale three-dimensional cell reconstructions. Shortly after outgrowth of the cell begins in Spizizen medium with glucose, the mesosome becomes an elongated structure in close contact with a rounded nucleoid. When nuclear replication reaches full activity, the mesosome develops into a single, complicated versatile system, with tubules that traverse the cytoplasm and have elaborations in and near the nucleoplasm. Later the system may retract to form large rounded mesosomes; the tubules and strings of vesicles within these mesosomes probably have been collected from the cytoplasm. Shortly after the first cell division, both sister cells have two nucleoids, but with longer generation times induced by growth in media containing acetate instead of glucose; these sister cells have only one nucleoid each. In acetate-grown cells rounded nucleoids that have no contact with a mesosome may represent nucleoids in a temporary stage of rest. On the other hand, the nucleoids of cells growing in glucose-containing medium are always penetrated by mesosomal material, superficially or deeply. Since the mesosome appears capable of traversing the nuclear fibrils, and even reaching the last strands connecting the dividing nucleoids, it is suggested that this organelle may play a vital role in the Bacillus division cycle.     

Ultrastructure of L-forms. II. L-forms of Listeria monocytogenes]

A study was made of ultrathin sections of the stable l-forms of listeria obtained under the action of penicillin in meat-peptone-liver broth. A marked cellular polymorphism was found in the L-form culture: within the same colony cells differed in size, shape and fine structure. It is supposed that polymorphism could be partially explained by a different plasticity and premeability of cytoplasmic membrane in different types of cells of the same L-colony. The three-layer structure of the membrane does not always display the same distinctness in various L-colony cells and also in different areas of the cell surface. Structureless material of low electron density, possibly a defective murein or its precursor, was revealed on the membrane surface. Electrondense inclusion bodies, mesosomes of ring-shaped or more complicated structure and two-contour vesicles were found in the cytoplasm. The cells multiplied by budding, by binary and anomalies division participation of mesosomes in this process was not proved by the L-forms.     

Fine structural observations of the peritubular cell layer in the hamster testis

Summary The seminiferous tubule of the hamster is surrounded by a layer of specialized cells. The fine structure of these cells is similar to that of smooth muscle. Their cytoplasm contains numerous fine filaments and dense areas; in addition, vesicles can be seen forming on the plasmalemma and are also free in the cytoplasm. It is possible that their contractions aid in the transport of spermatozoa through the seminiferous tubules.     

The DNA-structures of the chloroplast of Prorocentrum micans (Dinophyceae)

Summary DNA-regions of the chloroplasts of the dinoflagellate Prorocentrum micans were investigated by using serial sections. Prior to post-osmication the glutaraldehyde-fixed cells were treated with trypsin which results in a selective presentation of DNA-structures.For each of the two multilobed chloroplasts of the cell at least 80–100 individual DNA-regions could be calculated. Three-dimensional reconstructions of DNA-regions lead to models of usually flattened irregular discs which can differ markedly in size. It is concluded that the DNA-regions also differ in their DNA-content. Branched DNA-regions are regarded as possible division stages; they suggest a division into parts of different size.In some of the DNA-regions the DNA-fibrils seem to be attached to tube- or tongue-like evaginations of thylakoid membranes. The evaginations differ from normal thylakoids in their limited extension, enlarged loculus and their clearly visible unit membrane. A possible functional resemblance to bacterial mesosomes is discussed.Finally it is concluded that 1. the chloroplast of Prorocentrum is a polyenergidic organelle considering the number of DNA-regions, and that 2. the individual DNA-regions are polyploid to variable degrees with respect to their size.     

Electron microscopic demonstration and isolation of ribosomes in mesosomes from Staphylococcus aureus

Mesosomes of Staphylococcus aureus 209P were observed to be extruded as tubules upon protoplast formation by electron microscopy and isolated under hypertonic conditions to maintain their structural integrity by differential centrifugation followed by sucrose density gradient centrifugation. Isolated mesosomes were composed of long, branched tubules of irregular sizes and they were shortened during purification. Thin sections of isolated mesosomes showed that the mesosomal tubule was surrounded by a triple-layered membrane and contained ribosome-like particles in diameter of about 15 to 20 nm. These particles were isolated from purified mesosomal preparation by disrupting the mesosomal tubule with deoxycholate and Triton X-100 under hypotonic conditions followed by a linear sucrose density gradient centrifugation. Negatively stained preparations of the isolated particles revealed the same appearance as those of the ribosomes isolated from the cytoplasm. The mesosomal particles sedimented at 70S in sucrose gradients in the presence of 10 mM Mg2+, but they were dissociated into two subparticles, 50S and 30S subunits, upon lowering the Mg2+ concentration to 1 mM. These findings indicate that the mesosomal tubule is packed with ribosomes.     

Reversible changes in nuclear and cell surface topography in cells exposed to collagenase and EDTA

Summary Rabbit auricular chondrocytes, SIRC cells, human fibroblasts, and HeLa cells were cultivated in vitro and the fine structural effects of various detachment procedures studied. Treatment with collagenase, trypsin, and trypsin-EDTA caused scalloping of the nuclear envelope, accumulation of phagolysosomes, and an increase in the number of cell surface extensions. Collagenase-EDTA evoked a marked deformation of the nuclei with formation of numerous deep indentations and a redistribution of heterochromatin. Similarly, the cell surface became extensively folded and the vacuolation of the cytoplasm was further increased. These changes were reversible and within 24 h the cells had regained a normal structure. In all cases, chondrocytes and SIRC cells were most prominently affected, whereas fibroblasts and HeLa cells were only slightly changed. Treatment of chondrocytes with colchicine or cytochalasin B did not produce any effects of the type mentioned above. Neither did treatment with the drugs before and during detachment with collagenase-EDTA prevent the structural modification of the cells. It therefore seems unlikely that micro tubules and micro filaments are essential for this process. The structural changes occurring during detachment of cells could represent an adoptive mechanism for disposal of excessive membrane in connection with transition from a flattened to a rounded shape.Financial support was obtained from the Swedish Medical Research Council (proj.no. 12 x -03355), the King Gustaf V 80th Birthday Fund, the Swedish Society of Medical Sciences, the Jeansson Foundations, and the Bergvall Foundation. Expert technical assistance was provided by Karin Blomgren     

Role of lymphocyte surface determinants in lymph node homing

Thoracic duct lymphocytes briefly incubated in vitro with trypsin and then infused into syngeneic rats are unable to migrate into lymph nodes. Trypsin-treated lymphocytes incubated at 37 °C in the absence of enzyme for 12 hr recovered their lymph node homing properties. In vitro recovery did not occur if the cells were cultured at 17 °C. Evidence was obtained that trypsin cleaved sialyglycoproteins from the surface of lymphocytes and that these determinants reappeared after the cells were maintained at 37 °C for 24 hr.Puromycin added to cultures of normal lymphocytes for 3 hr before infusion markedly reduced the recovery of donor cells in lymph nodes. The results suggest that surface determinants of recirculating lymphocytes essential for homing into lymph nodes may be rapidly turned over.     

Estimation of protein digestibility—III. Studies on the digestive enzymes from the pyloric ceca of rainbow trout and salmon

Digestive proteinases were isolated and partially purified from the pyloric ceca of trout and salmon. Their stability and some catalytic properties were compared with those of a three-enzyme system that is used for determination of in vitro protein digestibility. In contrast to the three-enzyme system, pyloric ceca trypsin and total proteinase activity were least stable at pH values below 5.0 and most stable under alkaline conditions up to pH 10.0. Thermal inactivation (50%) occurred in 60 min at 55°C for trypsin activity of trout and salmon ceca proteinases and at 40°C for the three-enzyme system at the pH (8.0) of the in vitro assay. Thermal inactivation (50%) of total proteinase activity occurred in 60 min at about 55, 50 and 35°C for chinook, trout and three-enzyme preparations, respectively. SDS-PAGE zymograms of the ceca enzymes showed the presence of several proteolytic activity bands. Two of the bands corresponded in molecular weight to trypsin and chymotrypsin. Ceca proteinases differ from the three-enzyme system in their response to inhibitors; in particular, the ceca proteinases are much more sensitive to soybean trypsin inhibitor than the procine trypsin used in the three-enzyme system when assayed for trypsin, but less sensitive when assayed for total proteinase. The distinctive properties of ceca enzymes help explain why they are more appropriate than the three-enzyme system, and other enzyme cocktails for in vitro protein digestibility assay of saunonid feed components.     

Freeze-fracture of the normal and pathological megakaryocyte lineage in chronic megakaryocytic-granulocytic myelosis

Freeze-fracture and thin sections were performed on human bone marrow of chronic megakaryocytic-granulocytic myelosis (CMGM) to study the three-dimensional fine structure and maturation of normal and atypical megakaryocytes and thrombocytes. In the many normally maturing megakaryocytes the development of the demarcation membrane system (DMS) was best investigated by comparison of thin sections with freeze-fracture replicas. The DMS shows no connections with the Golgi apparatus or rough-surfaced endoplasmic reticulum, but originates from tubular infoldings of the plasma membrane. These infoldings are always in continuity with the extracellular space and form an intracellular membranous pool by branching and coalescing of flattened tubules from which finally the perforated cisternae of the DMS arise. Freeze-fracture of the normal thrombocytes confirms earlier findings. The abnormal giant platelets seen in CMGM display extensive areas of smooth membranes of a spongy structure consisting of dense tubules surrounded by the labyrinth of the surface-connected system. Their physiological significance in these atypical platelets remains unsolved.     

Regular Array in the Cell Wall of Lactobacillus fermenti as Revealed by Freeze-Etching and Negative Staining1

Freeze-etching of Lactobacillus fermenti F-4 (NCTC 7230) revealed that the outer layer of the cell wall was composed of a regular array in which parallel lines ran obliquely to the longitudinal axis of the cell with an average distance between the centers of about 9.6 nm and were intersected by thinner lines with an average periodicity of approximately 6.2 nm at an angle of about 75°. Occasionally the direction of the striation was discontinuously shifted near one end of the cell. Beneath the regular array the middle cell wall layer packed with granules and the smooth inner cell wall layer were discernible and the mesosomes were also visible in the cytoplasm. When the ultrastructure of isolated outer cell wall fragments was examined by negative staining, the regular array appeared to be composed of subunits, about 3.6 nm in diameter, which were arranged in a tetragonal pattern. The tetragonal array consisted of the subunits in rows in two directions at an angle of about 75° to each other. The average spacing between the rows was about 9.3 nm in one direction and 5.5 nm in the other direction.     

The fine structure of Herpetosiphon,and a note on the taxonomy of the genus

The fine structure of the Gram-negative filamentous gliding bacterium, Herpetosiphon is described. The outer membrane of the cell envelope could not be resolved as a separate structure, probably because it is fused with the underlying dense (peptidoglycan) layer. There was an additional wall layer outside this membrane-peptidoglycan complex, but a sheath in the classical sense, as postulated in the definition of the genus, was lacking. On the cell surface a loose network of fibrils could be seen. Inside the cells 3 types of intracytoplasmic membranes were discernible: a) true mesosomes near cross walls; b) a system of coarser membranes which was not connected with the septa and formed networks or tubular complexes; c) degenerated septa within bulbs. the bulbs are swollen sections of filaments, occurred mainly in ageing cultures, and are probably a degeneration phenomenon. The filaments contained necridia, i.e. dead and empty cells, across which breaks may occur so that empty cell wall cylinders remain attached to the ends of the daughter filaments, falsely suggesting the presence of a sheath. The taxonomy of Herpetosiphon is discussed in detail: The organism has been described before as Flexibacter giganteus. It is proposed to abandon the species H. aurantiacus in favor of H. giganteus, but to retain the genus Herpetosiphon. An improved definition of the genus is given.