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PubMed1983

Differences of asymmetrical division between the pseudomycelial and yeast forms of Candida albicans and their effect on multiplication.

Unlike asymmetrical division of budding yeast, the daughter cell in the pseudomycelial form of Candida albicans at division was nearly equal in size to the mother cell, it had a larger amount of protein, RNA and active protoplasm (cell size minus vacuolar volume) than the mother cell, and it budded earlier than the mother cell. Results presented here suggest that the cell size control over bud initiation found in budding yeasts is also applicable to the pseudomycelial cells of C. albicans if vacuolar volume is omitted from cell size.

Candida albicans↗
PubMed1979

Treponema succinifaciens sp. nov., an anaerobic spirochete from the swine intestine.

The morphology, the general physiological characteristics, and the energy-yielding metabolism of an obligately anaerobic spirochete isolated from the colon of a swine were studied. Electron microscopy showed that the helical spirochetal cells possessed an outer sheath, a protoplasmic cylinder, and 4 periplasmic fibrils in a 2-4-2 arrangement. The spirochete grew in an atmosphere of N2 in prereduced media containing a carbohydrate, NaHCO3, rumen fluid, yeast extract, peptone, L-cysteine, and inorganic salts. The spirochete fermented carbohydrates and required substrate amounts of CO2 (HCO3-) for growth. Amino acids were not fermented. Major fermentation products of cells growing with glucose as the substrate and in the presence of CO2 were acetate, formate, succinate, and lactate. Small amounts of 2,3-butanediol, pyruvate, and acetoin were also formed. Determinations of enzymatic activities in cell extracts, and of radioactivity in products formed by growing cells from [1-14C]glucose, indicated that this sugar was dissimilated to pyruvate via the Embden-Meyerhof pathway. The spirochetes used a coliform-type clastic reaction to metabolize pyruvate. Determinations of radioactivity in products formed from [14C]NaHCO3 indicated that CO2 was assimilated and used in succinate production. The guainine + cytosine content of the DNA was 36 mol %. This study indicates that this intestinal spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema succinifaciens.

Anaerobiosis↗
PubMed1980

Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria.

A saccharolytic spirochete that associated and interacted with cellulolytic bacteria was isolated from bovine rumen fluid. Isolation was accomplished by means of a procedure involving serial dilution of a sample of rumen fluid into a cellulose-containing agar medium. Clear zones appeared within the medium as a result of cellulose hydrolysis by rumen bacteria. The saccharolytic spirochete and a cellulolytic bacterium later identified as a strain of Bacteroides succinogenes were isolated from the clear zones. The spirochete did not utilize cellulose, but grew in coculture with the cellulotytic bacterium in cellulose-containing media. When cocultured in these media the spirochete used, as fermentable substrates, soluble sugars released from cellulose by the cellulolytic bacterium. In cellulose-containing agar medium the spirochete enhanced cellulose breakdown by the B. succinogenes strain. Electron microscopy showed that the helical spirochete cells possessed an outer sheath, a protoplasmic cylinder, and two periplasmic fibrils. Under a CO2 atmosphere, in a reduced medium containing inorganic salts, rumen fluid, glucose, and NaHCO3, the spirochete grew to a final density of 1.9 X 10(9) cells/ml. Succinate, acetate, and formate were products of the fermentation of glucose by growing cells. CO2 (HCO3-), branched short-chain fatty acids, folic acid, biotin, niacinamide, thiamine, pyridoxal, and a carbohydrate were required for growth of the spirochete. The results of this study indicated that the rumen spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema bryantii.

Animals↗
PubMed1982

Morphogenesis throughout saprobic and parasitic cycles of Coccidioides immitis.

The fungus, Coccidioides immitis, differs from other dimorphic pathogens in that its parasitic stage is a complex morphogenic cycle, raising the question that changes and composition during morphogenesis might influence host responses. As a prelude to examining the interaction of fungal morphogenesis and host responses, the life cycle of this fungus has been examined in greater detail than previously accomplished. During saprobic development, alternating enterothallic arthroconidia are formed as infectious propagules. The outer wall is broken and loosely adherent. Under in vitro conditions supporting the parasitic cycle, multinucleate arthroconidia transform into uninucleate round cells. Rapid, synchronous, nuclear replication is initiated, accompanied by increase in cell mass and deposition of new cell wall substance. As karyokinesis ceases, morphologic differentiation begins with invagination of the inner layers of the spherule wall and then is progressive, eventually segmenting the protoplasm into uninucleate endospores grouped in clusters within a hyaline membrane. Endospores, escaping through a break in the spherule wall, are held in aggregates by fibrils which are stretched and broken as endospores separate. It would seem that rapid production of hundreds of progeny from an original single cell, protected during development by an enclosing spherule wall and then released in clusters, should favor establishment of the fungus in a host, and dynamic changes in the cell wall during morphogenesis should influence the host response.

Cell Membrane↗
PubMed1976

A constitutive mutation in a posttranscriptional regulator gene for a phenoloxidase and proteases in Podospora anserina.

A recessive mutation in the gene mod-2 results in the synthesis at low temperatures of a phenoloxidase and an arrest of growth, reversible by beta-phenyl-pyruvic acid, a protease inhibitor. Phenoloxidase synthesis is 5-fluorouracil resistant and cycloheximade sensitive. Suppression of both cold sensitivity and phenoloxidase synthesis by common factors (higher NH4+ concentrations or mutations) suggests that the protease, suspected to be responsible for cold sensitivity, also arises from preexisting mRNA molecules. Instead of being recessive and constitutive, the mod-2 mutations is suppressive and dominant when cold sensitivity and phenoloxidases synthesis are induced as the consequence of the nonallelic gene interactions C/D, C/E, or R/V responsible for protoplasmic incompatibility. Combinations of nonallelic incompatibility systems and several mod-2 mutations lead us to hypothesize that the translational control of the above proteins depends on conformational relationships between incompatibility gene products and mod-2.

Ammonia↗
PubMed1988

The ultrastructure of the cysts of Blastocrithidia triatomae Cerisola et al. 1971 (Trypanosomatidae): a freeze-fracture study.

Cysts of Blastocrithidia triatomae from hosts and from in vitro cultures were investigated by freeze-fracture and freeze-etch electron microscopy. No differences in structure were observed between cysts frozen immediately and those fixed and cryoprotected with glycerol. The ultrastructure of all cysts differed considerably from that known for mastigote stages of trypanosomatids: the nuclear pores were surrounded by circular fracture-plane transitions between the inner and outer nuclear membranes; the matrix of the kinetoplast appeared banded; the mitochondrion and the endomembrane system were poorly developed; no flagellar apparatus or basal body could be found; and the subpellicular microtubules characteristic of trypanosomatids were absent. Beneath the surface membrane, a special region, 80-90 nm thick and consisting of several rows of closely packed particles was observed. Intramembrane particle (IMP) distribution on the fracture faces of the surface membrane was highly asymmetric: the ectoplasmic face had very few IMPs, whereas the protoplasmic face contained numerous, closely packed IMPs. No external cyst wall was present.

Animals↗
PubMed1980

A new staining method of astrocytes for paraffin section.

A new method of staining astrocytes in formation-fixed, paraffin-embedded sections was devised: (1) fix them in 5% mercuric chloride solution for 30 min to 1 h at 56 degrees C, (2) then place in 0.5% iodine alcohol for 5 min followed by placing in 0.5% sodium thiosulfate for 5 min, (3) immerse in 0.25% potassium permanganate for 3 min, (4) place in 2% oxalic acid for 2 min. (5) mordant in 2% iron alum for 45 s, and (6) place in 2% silver nitrate solution for 30 min. The next step is impregnation in ammoniacal silver solution for 10 -- 15 min at 56 degrees C, followed by reduction in neutral formalin and 2% iron alum, toning in 0.2% gold chloride, and fixing in 5% sodium thiosulfate. Pathological astrocytes of fibrillary and protoplasmic types were distincly demonstrated, although nerve cells and nuclei of oligodendrocytes and microglial cells were also faintly stained. Thus, the staining for paraffin sections is fairly selective for astrocytes in pathological states.

Astrocytes↗
PubMed1984

Crystallization of intramembrane particles in rabbit sarcoplasmic reticulum vesicles by vanadate.

Sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle appear in freeze-fracture as 0.15-0.2 micron vesicles. The concave fracture surface (P-face) contains a dense population of 8.5 nm particles that were previously identified as the Ca2+-transport ATPase. The convex surface (E-face) is mostly smooth, displaying an occasional particle but no complementary arrays of pits. Incubation of the vesicles at 4 degrees C in calcium-free solutions containing 5 mM Na3VO4 induces the formation of two-dimensional crystalline arrays of the Ca2+, Mg2+-ATPase, accompanied by structural changes visible by freeze-etch electron microscopy. Most vesicles elongate into tubules 60-80 nm in diameter and the 8.5 nm intramembrane particles of the P-face become regularly organized into parallel ridges. The ridges are coiled around the tubules in right-handed helices, oriented at 50-60 degrees angle to the long axis of the tubules. The particles repeat along the rows at about 5.5 nm and the rows repeat at 10.5-11.0. Occasionally the ridges seem to break up into 8.5 nm particles. Parallel furrows are visible on the (convex) E-face of the tubules. In high resolution replicas, the furrows are resolved into rows of pits that are complementary images of the ridges. Deep etching and rotary shadowing reveal oblique crests on the protoplasmic surface, consisting of dimeric particles close to 8.5 X 5.5 nm in size, in which each monomer can frequently be resolved into two structural domains. These data suggest that vanadate induces a conformational change in the Ca2+-transport ATPase, with crystallization of the intramembrane particles.

Animals↗
PubMed1986

The surface membrane of Leishmania mexicana mexicana: comparison of amastigote and promastigote using freeze-fracture cytochemistry.

The freeze fracture replica technique has been used to compare the plasma membranes of amastigote and promastigote stages of Leishmania mexicana mexicana with respect to intramembranous particle (integral protein) distribution and to beta-hydroxysterols content as revealed by the distribution of lesions induced by the polyene antibiotic filipin. Intramembranous particle (IMP) density was greater in promastigote than in amastigote plasma membranes. Intramembranous particles were more abundant in the protoplasmic face (PF) than in the exoplasmic face (EF) of promastigotes, but this situation was found to be reversed in amastigotes. Filipin-induced lesions in glutaraldehyde-fixed parasites indicated higher levels of beta-hydroxysterols in the amastigote than in the promastigote plasma membrane, and in the promastigote flagellar membrane than in the body membrane. Amphotericin B (a related polyene antibiotic used in chemotherapy of leishmaniasis) induced IMP aggregation in the PF of unfixed amastigotes but did not appear to influence sterol distribution as demonstrated by freeze-fracture of subsequently-fixed and filipin-treated organisms.

Amphotericin B↗
PubMed1995

Morpho-functional modifications of human syncytiotrophoblast plasma membrane during pregnancy induced hypertension.

A decrease of Na+/K(+)-ATPase activity has been reported in syncytiotrophoblast plasma membrane (SPM) obtained from pregnancy induced hypertensive (PIH) women. The aim of the present work was to verify if the reported modifications in activity are due to a decreased number of enzymatic molecules or to a conformational change of the enzyme itself. Morphological studies were performed in order to better understand the relations between the enzymatic protein and the lipid bilayer. Kinetic studies were also performed. SPM obtained from PIH showed: i) an increased affinity of Na+/K(+)-ATPase for ouabain binding, ii) a significant change in the maximum velocity of the enzyme, iii) a higher distribution factor (DF) of intramembrane particles (IMPs) in the exoplasmic face of the membrane, iv) a decreased mean diameter of IMPs both in the protoplasmic and exoplasmic faces, v) a decreased number of IMPs in the exoplasmic face. In conclusion, a conformational modification seems to be at the basis of the decreased Na+/K(+)-ATPase activity during PIH as suggested by binding, ultrastructural and kinetic data herein reported.

Adult↗
PubMed1984

A4: an antigenic marker of neural tube-derived cells.

The A4 monoclonal antibody was originally found to bind to the surface of the majority of neurons in rat CNS cultures, but not to PNS neurons or non-neural cells. It was subsequently shown to bind to immature oligodendrocytes and their precursor cells but not to the most mature oligodendrocytes. In the present study, we have used immunofluorescence assays on cell suspensions and cultures and on semi-thin, frozen tissue sections to show that protoplasmic and fibrous astrocytes and most ependymal cells are also A4+. Taken together, these results suggest that in adult rats A4 is expressed exclusively by cells of the CNS and that all cell types derived from the neural tube are A4+, at least at some time in their development. While neurons, astrocytes and ependymal cells continue to express the antigen in adults, most oligodendrocytes appear to lose it as they mature. The finding that macrophages in CNS cell suspensions and cultures are A4- suggests that microglial cells are not derived from the neural tube.

Animals↗
PubMed1984

Freeze-fracture morphology of the vestibular hair cell--primary afferent synapse in the chick.

The intramembrane specializations at vestibular hair cell-primary afferent synapses have been identified and characterized in complementary freeze-fracture replicas from prehatch and hatchling chick cristae and maculae. Hair cell protoplasmic (P) faces at sites where presynaptic bodies are present exhibit small, tightly packed arrays of 9 nm particles. Hair cell external (E) faces have corresponding arrays of pits. Multiple arrays are often observed in contiguity. Opposite the presynaptic bodies, postsynaptic afferent boutons and calyces exhibit a more extensive array of scattered, irregular E-face particles. Corresponding P-fracture faces of afferent boutons and calyces display little topographical specialization opposite these E-face arrays, which are presumed to be the intramembrane correlate of the postsynaptic density. Examination of complementary replicas has allowed identification of the intramembrane synaptic specializations for all membrane faces at the synaptic apposition.

Animals↗
PubMed1984

Freeze-fracture of sensory nerve endings in cat muscle spindles.

Sensory nerve endings and intrafusal muscle fibres of cat muscle spindles were examined by freeze-fracture electron microscopy. The purpose of the study was to reveal intramembranous specializations that might be associated with the mechanosensory transducer system. Nerve ending membranes were found to contain high concentrations of intramembranous particles (greater than 7500 per micron2) in a range of sizes, but no localized aggregations or non-random arrangements were observed. Nerve ending membranes directly apposed to the surfaces of intrafusal muscle fibres did not appear to be strikingly different from non-interface membranes, although some differences in particle concentrations could be detected. The membranes of endings on nuclear bag and nuclear chain muscle fibres differed slightly in the numbers of particles and their distributions to protoplasmic or external fracture faces. It was concluded that intramembranous elements of the mechanoelectric transducer system may be dispersed over the whole surface of a sensory ending, rather than localized to smaller domains.

Animals↗
PubMed1982

Synaptic vesicle activity in stimulated and unstimulated photoreceptor axons in the housefly. A freeze-fracture study.

Light-stimulated and unstimulated photoreceptor (retinular) axon terminals in the lamina ganglionaris (first optic neuropil) of the housefly are examined using freeze-fracture replication. The presence of numerous, cross-fractured capitate projections permits unmistakable identification of the retinular axon terminal membrane. Regardless of the conditions of illumination, the protoplasmic face (P-face) of the terminal membrane contains numerous bowtie-shaped particle clusters (active zones) which resemble the en face form and disposition of the presynaptic ribbon found in thin sections. Estimates from freeze-fractured material indicate that each retinular axon possesses at least 175 such active zones. In eyes fixed during illumination, active zones are surrounded by many membrane dimples indicative of vesicle fusion sites. Such synaptic vesicle sites are seldom encountered in terminals which are dark-adapted and fixed in the dark. Results from light-adapted eyes placed in the dark following the onset of fixation suggest that endocytosis may occur in the extrasynaptic regions of this inhibitory synapse. P-face particles are uniformly distributed throughout the extrasynaptic regions of unstimulated terminals. Particle density increases in areas peripheral to the active zones in stimulated eyes, particularly within the regions presumed to be undergoing active endocytosis. These structural findings are discussed in the context of the Heuser-Reese model of vesicle exocytosis and recycling.

Animals↗
PubMed1993

Distribution of glial fibrillary acidic protein and glutamine synthetase in human cerebral cortical astrocytes--a light and electron microscopic study.

Human cerebral cortex was studied immunocytochemically by light and electron microscopy using antibodies against glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). Glial fibrillary acidic protein-positive cells and processes were present in both cortex and white matter, but in contrast glutamine synthetase-positive cells and processes were present only in cortex. Cell bodies which contained glutamine synthetase had typical ultrastructural features of protoplasmic astrocytes. Glutamine synthetase-positive processes were often present near asymmetrical synapses in the neuropil. These processes often contained mitochondria, but not glial filaments, and were different from unlabelled astrocytic processes, which seldom contained mitochondria, but had large numbers of glial filaments. Glutamine synthetase immunoreactivity therefore affords a means of distinguishing between these two types of astrocytic processes in the human cerebral cortex.

Adult↗
PubMed1990

Macromolecular structure of axon membrane and action potential conduction in myelin deficient and myelin deficient heterozygote rat optic nerves.

The macromolecular structure of the axon membrane in optic nerves from 25-day-old male littermate control and myelin deficient (md) rats and 16-month-old md heterozygotic rats was examined with quantitative freeze-fracture electron microscopy. The axon membrane of control optic nerves displayed an asymmetrical partitioning of intramembranous particles (IMPs); P-fracture faces of myelinated internodal axon membrane were more particulate than those of pre-myelinated axons (approximately 1600 v 1100 microns-2, respectively), while relatively few IMPs (approximately 150 microns-2) were present on external faces (E-faces) of internodal or pre-myelinated axon membrane. Amyelinated axons of md optic nerves also exhibited an asymmetrical partitioning of IMPs; protoplasmic membrane face (P-face) IMP densities, taken as a group, exhibited a wide range (approximately 600-2300 microns-2) and, in most regions, E-faces displayed a relatively low IMP density (approximately 175 microns-2). Axons of greater than 0.4 microns diameter exhibited significantly greater mean P-face IMP density than axons less than 0.4 microns diameter. Aggregations of E-face IMPs (approximately 350 microns-2) were occasionally observed along amyelinated axon membrane from md optic nerves. Optic nerves from md heterozygote rats exhibit myelin mosaicism, permitting examination of myelinated and amyelinated axon membrane along the same tract. The axon membrane exhibits different ultrastructure in these two domains. Myelinated internodal axon membrane from md heterozygote optic nerves exhibits similar P- and E-face IMP densities to those of control internodal axolemma (approximately 1800 and 140 microns-2, respectively). Amyelinated axons in the heterozygote exhibit a membrane structure similar to amyelinated axons in md optic nerve. P-face IMP density of large diameter (greater than 0.4 microns) amyelinated axons from md heterozygote optic nerves is significantly greater than that of small calibre (less than 0.4 microns) axons. In most regions, amyelinated axon membrane exhibits a relatively low E-face IMP density (approximately 200 microns-2); however, focal aggregations (approximately 400 microns-2) of E-face particles are present. Electrophysiological recordings demonstrate that amyelinated axons in md optic nerves support the conduction of action potentials. Compound action potentials in md optic nerves exhibit a monophasic configuration, even at 20-days postnatal, similar to that of pre-myelinated optic nerve of 7-day-old normal rats. Moreover, conduction velocities in the amyelinated 20-day-old md optic nerve are similar to those displayed by pre-myelinated axons from 7-day-old optic nerves.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1979

Molecular specializations of the axon membrane at nodes of Ranvier are not dependent upon myelination.

Nodes of Ranvier from normal and 'dystrophic' mice have been examined with quantitative freeze-fracture electron microscopy. Regions of nodal, paranodal and interparanodal axolemma of normal fibres are clearly distinguishable on the basis of particle size distributions in electron micrographs of freeze-fractured replicas. Protoplasmic fracture faces of normal nodes of Ranvier, contain approximately 40% 100 A particles and about 25% elongated particles 150 by 250 A. Paranodal and interparanodal membranes contain a more uniform distribution of smaller diameter particles. 'Dystrophic', mice of the 129/ReJ-Dy strain have a genetic defect of Schwann cell development and myelinogenesis. Axons of the sciatic and deep peroneal nerves in dystrophic mice, which appear to be normally myelinated, possess approximately the same distributions of particles as axons in normal mice. However, in affected regions of the ventral and dorsal roots, Schwann cell wrappings may be missing, creating heminodes of Ranvier where the myelination terminates or begins again. At such heminodes, there is a circular band of axonal membrane which bears particles of sizes and packing densities similar to that found at normal nodes. High voltage electron microscopic examination of 0.25--1 micron thick sections from these hemi-nodal regions reveals the presence of a filamentous layer beneath the particle-rich membrane. In addition, completely amyelinated regions of root axons contain particle patches having size-density distributions similar to that of both normal and hemi-nodal membranes. Thus, the nodal membrane displays a characteristic particle-size distribution profile. The occurrence of this particle profile does not appear to be dependent upon the presence or absence of Schwann cells. These observations suggest that the functions subserved by the numerous particles at the node of Ranvier are not dependent upon myelination for their local differentiation within the axonal membrane.

Animals↗
PubMed1993

Differentiation of Dunn osteosarcoma cells in response to dibutyryl cyclic 3',5'-adenosine monophosphate.

We investigated the effects of dibutyryl cyclic adenosine 3',5'-monophosphate (Bt2cAMP) on the differentiation of Dunn osteosarcoma cells. Flow-cytometric analysis and DNA synthesis assay showed that Bt2cAMP decreased the cell population in the S phase in a time- and dose-dependent manner. Also, the cells showed distinct morphological and functional alterations; the cell morphology changed to a fibroblast-like appearance with long and thin protoplasmic processes, the knobs or blebs on both the cell membrane and nuclear membrane disappeared and the intracellular alkaline phosphatase activity increased. Moreover, Bt2cAMP-treated cells secreted a large quantity of fibronectin, which was deposited on the extended cell surface in the culture medium. Thus, Dunn osteosarcoma cells are differentiated morphologically and functionally by Bt2cAMP, and might be transformed to benign precursor cells.

Alkaline Phosphatase↗