Biomedical subjects

E M Schraner

Publications and source records attributed to E M Schraner.

17 recordsLinked to original sources

PubMed2001

Enhanced resolution of membranes in cultured cells by cryoimmobilization and freeze-substitution.

Investigations of cellular processes demand immediate arresting of the process at any given time and excellent retention of cellular material and excellent visibility of membranes. To achieve this goal we used cryofixation to arrest cellular processes instantly and tested diverse freeze-substitution protocols. Madin-Darby kidney cells and Vero cells were grown on carbon-coated sapphire disks. For cryofixation the sapphire disks covered with a cell monolayer were injected with the aid of a guillotine into liquid propane or ethane or a mixture of both cooled by liquid nitrogen. Freezing of the cryogen was prevented by using a partially insulated cylinder and by vigorous stirring that results in a substantial decrement of the freezing point of the cryogen. Cell monolayers can be cryofixed successfully using the guillotine in a safety hood at ambient temperature and humidity or at 37 degrees C and 45% humidity. The freezing unit can also be placed in a laminar flow for working under biohazard conditions. For visualizing cell membranes at high contrast and high resolution, cells were substituted in the presence of various concentrations of glutaraldehyde and osmium tetroxide and the temperature was raised to diverse final temperatures. Substitution for 4 hours at -90 degrees C in anhydrous acetone containing 0.25% anhydrous glutaraldehyde and 0.5% osmium tetroxide followed by a temperature rise of 5 degrees C/hour to 0 degrees C and final incubation for 1 hour at 0 degrees C resulted in high contrast and excellent visibility of subcellular components at the level of the membrane bilayer. The high spatial and temporal resolution makes this methodology an excellent tool for studying cell membrane-bound processes, such as virus-cell interactions.

Animals↗

PubMed2000

Structural analysis of F18 fimbriae expressed by porcine toxigenic Escherichia coli.

The F18 fimbriae expressed by porcine toxigenic Escherichia coli strains are 1- to 2-mm-long filaments that mediate the adhesion of the bacteria to enterocytes. The backbone of these fimbriae is built from a major structural 15.1-kDa protein, FedA. The structure of isolated negatively stained F18 fimbriae imaged by dark-field scanning transmission electron microscopy (STEM) was resolved to approximately 2 nm. Analyzing their helical symmetry showed the axially repeating units to alternate in a "zigzag" manner around the helical axis with an axial rise of 2.2 nm. Two repeating units give rise to the observed 4.3-nm helical repeat, which is practically identical to the pitch of the one-start helix formed. Additionally, an axially repeating pattern with a 27-nm spacing was found on rotary-shadowed fimbriae. Mass-per-length determination of unstained F18 fimbriae by STEM revealed the axially repeating unit to have a molecular mass of 25.4 kDa, indicating that it is a FedA monomer, with the difference in mass arising from the minor subunits, FedE and FedF. The presence of the latter two proteins might cause the observed 27-nm axial pattern.

Animals↗

PubMed1998

Novel entry pathway of bovine herpesvirus 1 and 5.

Herpesviruses enter cells by a yet poorly understood mechanism. We visualized the crucial steps of the entry pathway of bovine herpesvirus 1 (BHV-1) and BHV-5 by transmission and scanning electron microscopy, employing cryotechniques that include time monitoring, ultrarapid freezing, and freeze substitution of cultured cells inoculated with virus. A key step in the entry pathway of both BHV-1 and BHV-5 is a unique fusion of the outer phospholipid layer of the viral envelope with the inner layer of the plasma membrane and vice versa resulting in "crossing" of the fused membranes and in partial insertion of the viral envelope into the plasma membrane. The fusion area is proposed to function as an axis for driving the virus particle into an invagination that is concomitantly formed close to the fusion site. The virus particle enters the cytoplasm through the opened tip of the invagination, and the viral envelope defuses from the plasma membrane. There is strong evidence that the intact virus particle is then transported to the nuclear region.

Animals↗

PubMed1997

Reevaluation of the effect of lysoyzme on Escherichia coli employing ultrarapid freezing followed by cryoelectronmicroscopy or freeze substitution.

Lysozyme is able to lyse Gram-positive bacteria acting as muramidase on the peptidoglycan polymer. Gram-negative bacteria in vitro are not lysed by lysozyme. It was assumed that the peptido-glycan is protected by the outer membrane and thus that Gram-negative bacteria are not affected by lysozyme without the aid of other factors such as EDTA or complement which enable lysozyme to penetrate the outer membrane. Accidentally, Pellegrini et al. [(1992) J. Appl. Bacteriol., 72:180-187] found that lysozyme per se is able to kill some Gram-negative bacteria. On the basis of morphological and immunocytochemical findings obtained from chemically fixed bacteria, it was concluded that lysozyme does not lyse Gram-negative bacteria but affects the cytoplasm of for example, Escherichia coli, leading to its disintegration, whilst the membranes do not break down. In an attempt to clarify the action of lysozyme on E. coli, we employed cryotechniques including ultrarapid freezing, cryomicroscopy and freeze substitution, and immunolabeling. Bacteria that were immediately frozen after exposure to lysozyme remained morphologically intact. Individual bacteria plated on agar after exposure to lysozyme were mostly intact when frozen within a few seconds. However, inner and outer membranes of 80% of the bacteria were disrupted, whereas the cytoplasm of only a few bacteria showed signs of disintegration when bacteria were frozen with a delay of only 5 min of plating onto pure agar or agar containing growth medium. After a period of time of 15 min between plating onto agar and freezing, about 97% of the bacteria showed changes of disintegration of various extent. Immunolabeling showed that lysozyme binds to the outer cell membrane and may penetrate the membrane, reaching the periplasmic space and possibly the inner cell membrane. The ultrastructural findings and the results of antibacterial assays suggest that lysozyme is bactericidal for E. coli but is not able to induce disintegration. Disintegration is accomplished by changes of the environment starting at the cell membranes. The mechanism by which lysozyme penetrates the membrane, the way it acts to be bactericidal, and the way disintegration is initiated remain to be clarified.

Escherichia coli↗

PubMed1993

Morphometric analysis of parathyroids in hypophysectomized rats in normocalcaemic and short-term induced hypocalcaemic conditions.

The ultrastructure of parathyroid glands of hypophysectomized rats in normo- and hypocalcaemic conditions was qualitatively and quantitatively analysed. Parathyroid glands of both intact and hypophysectomized rats comprised cells of similar shape and staining intensity and of an equal number of cells in mitosis. Cell volume was not affected but the surface area of plasma membrane, RER and Golgi complex was increased in parathyroid cells after hypophysectomy. Parathyroid cells of both intact and hypophysectomized rats responded to hypocalcaemia by a drastic increase in surface area of the plasma membrane and a decrease in surface area of the Golgi complex. Since RER, Golgi complex and plasma membrane are concerned with secretion of parathyroid hormone, the data suggest that (1) the pituitary has either no effect on parathyroid cells, or a possibly stimulating effect can be compensated after hypophysectomy, or the pituitary even has a suppressive effect on parathyroid cells, and (2) the responsiveness of parathyroid cells to changes in serum calcium is unimpaired after hypophysectomy.

Animals↗

PubMed1992

Cellular changes in rat parathyroids provoked by progesterone and testosterone.

Male rats kept on a standard diet were treated either with progesterone or testosterone by a single intramuscular injection of preparations which are slowly absorbed and metabolized. The rats were anaesthetized 24 h after application of the hormones, perfused with glutaraldehyde, and the parathyroid glands prepared for electron microscopy. Morphometric analysis revealed that both progesterone and testosterone provoked (1) an increment in nuclear and cell volume and a concomitant increment in cell surface area, and (2) an increment in surface area of rough endoplasmic reticulum by 42% and 49%, and of the Golgi complex by 85% and 63%, respectively. Previously, we had found that oestradiol treatment led to a similar response in parathyroid cells. The conclusion is thus drawn that male and female sex hormones induce membrane synthesis resulting in an enhanced capacity for parathyroid hormone secretion since RER and Golgi complex are concerned with this secretion. It is considered probable that sex hormones have the ability fundamentally to modulate secretory activity in parathyroid cells.

Animals↗

PubMed1991

Morphometric analysis of compartments concerned with secretion of parathyroid hormone in male and female rats.

Parathyroid cells responded to estradiol application in rats by increment in surface area of the compartments concerned with parathyroid hormone secretion, i.e. the rough endoplasmic reticulum, Golgi complex and secretory granules. We thus compared the morphology of parathyroids of male rats with those of females of the same age and same weight, respectively. Morphometric analysis revealed that (i) the mean cell volume was significantly larger and, consequently, the number of parathyroid cells lower expressed per 1 mm3 tissue and (ii) the surface area of the Golgi complex was larger expressed per mean parathyroid cell volume and per 1 mm3 tissue in female rats than in males. Diversity in cell volume seems to depend on age rather than on sex, whereas diversity in the Golgi complex is considered to be sex related. The physiological implications remain to be clarified.

Aging↗

PubMed1990

Parathyroid cell polarity as revealed by cytochemical localization of ATPases, alkaline phosphatase and 5'-nucleotidase.

Activities of Ca2(+)-dependent ATPase, Mg2(+)-dependent ATPase, Na(+)-K(+)-dependent ATP-ase, alkaline phosphatase, and 5'-nucleotidase were demonstrated after incubation of 40-microns vibratome sections of bovine parathyroids and subsequent visualization by electron microscopy. Prior to sectioning, parathyroid tissue was fixed with 1% glutaraldehyde for localization of alkaline phosphatase, and with 2% formaldehyde and 1% glutaraldehyde for demonstration activities of ATPases and 5'-nucleotidase. The activities of the five enzymes were found at the apicolateral domain of the plasma membrane in parathyroid cells, i.e. at the site parathyroid cells face neighbouring parenchymal cells. Ca2(+)-ATPase activity was also seen on mitochondria, Golgi complex and RER. The presence of these plasma membrane associated enzymes at the apicolateral domain only indicate polarity in parathyroid cells. It further suggests that many processes including transmembrane transport take place at the apicolateral domain, the site of parathyroid cells opposing blood capillaries.

5'-Nucleotidase↗

PubMed1990

Adhesive fimbriae produced in vivo by Escherichia coli O139:K12(B):H1 associated with enterotoxaemia in pigs.

Two strains of E. coli O139:K12 (B):H1 were compared in vitro and in the intestinal environment. Both strains colonized the small intestines of experimentally inoculated pigs and exhibited in vivo a similar relationship to the microvillus border as enterotoxigenic E. coli (ETEC). Strain 107/86 grown on blood agar expressed numerous long flexible non-haemagglutinating fimbriae which were antigenically distinct from the known fimbriae of porcine ETEC. It adhered in vitro to porcine enterocyte brush border fragments. Strain 124/76 grown on blood agar was devoid of fimbriae and did not adhere to brush border fragments. However, fimbriae morphologically and antigenically indistinguishable from those of strain 107/86 were detected in the intestinal environment by direct immunofluorescence and by immuno electron microscopy.

Animals↗

PubMed1989

Effects of estradiol on parathyroid cell activity.

Beta-estradiol-3-benzoate provoked an initial centrifugal membrane shift in rat parathyroid cells and, later, enlargement of the compartments concerned with parathyroid hormone secretion, which suggests that estradiol modulates not only transport and release of parathyroid hormone but also the capacity for its synthesis, packaging and storage.

Animals↗

PubMed1989

Potency of microwave irradiation during fixation for electron microscopy.

Liver, skeletal muscle, peripheral nerves, pancreas, thyroid and adrenal cortex were prepared for electron microscopy employing microwave energy either during prefixation with glutaraldehyde or instead of prefixation. Microwave irradiation in the presence of glutaraldehyde in Na/K-phosphate or Na-cacodylate containing CaCl2 and MgCl2 led to distinct appearance of membranes, mainly plasma membrane, and membranes of SER, Golgi complex and mitochondria in liver, pancreas and muscle. The area of high quality fixation, however, was limited to the periphery of samples. On the other hand, SER was dilated in cells of the adrenal cortex, and RER markedly vacuolated in thyroid follicular cells. Microwave irradiation in the presence of Na/K-phosphate and subsequent osmication resulted in preservation of the ultrastructure in similar quality as was obtained by osmication without previous immersion in glutaraldehyde. However, the preservation of SER and Golgi complex in liver and pancreas, and of mitochondria in muscle was greatly improved. Small myelin sheaths remained intact whereas large ones showed focal disintegration. We consider that enhancement of fixation by microwave energy may greatly improve preservation of membranes in some tissues. Successful fixation depends on the use of glutaraldehyde during microwave irradiation, the type of buffer, the addition of ions to increase stabilization, the exposure time to heat, and on postosmication.

Adrenal Cortex↗

PubMed1989

Quantitative assessment of cellular changes provoked by microwave enhanced fixation of parathyroids.

Rat parathyroids fixed by microwave enhancement, i.e. microwave irradiation in the presence of glutaraldehyde for 8 s and postfixation with OsO4 after a delay of 5 min, were compared with parathyroids fixed by perfusion with glutaraldehyde followed by immersion in glutaraldehyde and finally in OsO4. Morphometric analysis revealed that microwave enhanced fixation led to a larger mean cell volume, to larger cell surface area, and to larger surface area in membranes of RER and secretory granules. Though it is not known by which method parathyroid cells are conserved closer to the living state it is obvious that microwave enhanced fixation retains more membranes but provokes centrifugal dislocation of membranes mimicking exocytosis.

Animals↗

PubMed1988

Unimpaired membrane dynamics in parathyroid cells in insulin deficient rats.

The responsiveness of parathyroid cells in insulin deficient and short-term diabetic rats was investigated by morphometric analysis. Insulin deficiency was produced by intravenous injection of D-mannoheptulose and short-term (7 days) diabetes mellitus by intraperitoneal application of streptozotocin. Parathyroid glands were stimulated for parathyroid hormone secretion by decreasing the serum calcium concentration through intravenous infusion of EGTA. Parathyroid cells of controls, insulin deficient, and short-term diabetic rats responded to reduced serum calcium by a 45% increase of the cell surface area. This increase is assumed to be the result of the membrane-bound transport of parathyroid hormone from the Golgi complex and secretory granules to the plasma membrane and subsequent exocytic release of parathyroid hormone induced by the low serum calcium concentration. Therefore, the unimpaired increase in the cell surface area of parathyroid cells in insulin deficient and short-term diabetic rats indicates that insulin does not modulate the release of parathyroid hormone. It is also considered likely that synthesis of parathyroid hormone is not suppressed in short-term diabetes but that fat metabolism is disturbed leading to accumulation of lipid vacuoles.

Animals↗

PubMed1987

Parathyroid cell variants may be provoked during immersion fixation.

Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO4 in the same buffers or, alternatively, in s-collidine. Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5-2% formaldehyde and 2.5-5% acrolein in 0.1 M Na-cacodylate with or without Ca2+ and Mg2+, Na-phosphate or Na/K-phosphate at 4 degrees C followed by postfixation with 1% OsO4 in the same buffers or in s-collidine containing sucrose, Ca2+ and Mg2+. This procedure largely abolished the occurrence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.

Aldehydes↗

PubMed1987

Parathyroid ultrastructure after aldehyde fixation, high-pressure freezing, or microwave irradiation.

Parathyroid cell variants, commonly observed in parathyroid glands fixed by immersion in glutaraldehyde, are believed to be the result of cyclic changes in the course of parathyroid hormone secretion. Immersion of bovine parathyroid glands in a mixture consisting of 1% glutaraldehyde, 1.5% formaldehyde, and 2.5% acrolein, followed by post-fixation in 1% osmium tetroxide, resulted in high uniformity with only one cell variant, whereas the same fixation procedure led to disruption of cell membranes and formation of cell variants in rat parathyroids. Parathyroid glands of both cattle and rats prepared by high-pressure quick-freezing and subsequent freeze-substitution contained only one cell variant. Excellent preservation of the ultrastructure of bovine and rat parathyroids, also exhibiting only one cell variant, was achieved by microwave irradiation in the presence of 2.5% glutaraldehyde in Na-cacodylate followed by post-fixation with OsO4 in Na-cacodylate or s-collidine, both containing Ca2+ and Mg2+. Use of the appropriate buffer, as well as osmication, is essential for successful fixation utilizing microwave energy. The main effects are considered to be heating specimens within sufficient short periods and enhancement of subsequent osmium fixation. The results support the idea, arising after examination of perfusion-fixed parathyroid tissue, that parathyroid cell variants occur during improper aldehyde fixation rather than that they express functional diversity.

Air Pressure↗

PubMed1986

Ultrastructural alterations in mammalian parathyroid glands induced by fixation.

The influence of fixation methods, buffers and ions on the ultrastructure of parathyroid cells was studied in dogs, cats, rats and mice. Parathyroids fixed by immersion showed 3 chief cell variants referred to as cells in active, intermediate and resting stages, multinucleated syncytial cells, atrophic cells and, only in 1 feline parathyroid, a few oxyphil cells. Parathyroid glands fixed by perfusion, however, consisted only of 1 cell type. Satisfactory preservation was achieved by perfusion with 2.5% glutaraldehyde in 0.1 M Na cacodylate containing 0.25 mM CaCl2 and 0.5 mM MgCl2, and postfixation with 1% OsO4 in 0.1 M s-collidine containing 0.5 mM CaCl2 and 1.0 mM MgCl2. Good preservation was also obtained using Na phosphate during prefixation and postfixation. Other combinations of buffers led to shrinkage, dilation of rough endoplasmic reticulum cisternae, disruption of membranes or loss of matrix and secretory granules. The results demonstrate that the variants of parathyroid chief cells, multinucleated syncytial cells and atrophic cells arise during fixation.

Animals↗

PubMed1984

Quantitative aspects of membrane shifts in rat parathyroid cells initiated by decrease in serum calcium.

The secretory activity of parathyroid glands in rats was stimulated by decreasing the serum Ca++ concentration through constant intravenous infusion of EGTA. The morphometric analysis of the nuclear and cytoplasmic volume and of the surface area of the rough endoplasmic reticulum, Golgi complex, secretory granules and plasma membrane revealed a membrane shift from secretory granules and Golgi complex to the plasma membrane within 1 hr of calcium depression. Subsequently, between 1 and 3 hr of calcium depression, the membrane shift was from the plasma membrane to the Golgi complex. It is considered likely that these membrane shifts are related to a rise in release of parathyroid hormone by exocytosis and a subsequent increase in retrieval of plasma membrane by endocytosis--probably through the compartment of coated pits and coated and uncoated vesicles.

Animals↗