Evaluation of Structural Changes Induced by High Hydrostatic Pressure in Leuconostoc mesenteroides

Scanning electron microcopy (SEM), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC) were used to evaluate structural changes in Leuconostoc mesenteroides cells as a function of high-hydrostatic-pressure treatment. This bacterium usually grows in chains of cells, which were increasingly dechained at elevated pressures. High-pressure treatments at 250 and 500 MPa also caused changes in the external surface and internal structure of cells. Dechaining and blister formation on the surface of cells increased with pressure, as observed in SEM micrographs. TEM studies showed that cytoplasmic components of the cells were affected by high-pressure treatment. DSC studies of whole cells showed increasing denaturation of ribosomes with pressure, in keeping with dense compacted regions in the cytoplasm of pressure-treated cells observed in TEM micrographs. Apparent reduction of intact ribosomes observed in DSC thermograms was related to the reduction in number of viable cells. The results indicate that inactivation of L. mesenteroides cells is mainly due to ribosomal denaturation observed as a reduction of the corresponding peak in DSC thermograms and condensed interior regions of cytoplasm in TEM micrographs.     

In vivo characterization of thermal stabilities of Aeropyrum pernix cellular components by differential scanning calorimetry

Revival studies of Aeropyrum pernix show that the viability of cells and cell recovery after heat treatment depends on the temperature of treatment. Differential scanning calorimetry (DSC) is used to analyze the relative thermal stabilities of cellular components of A. pernix and to identify the cellular components responsible for the observed lag phase and reduced maximum growth following a heat treatment. DSC thermograms show 5 visible endothermic transitions with 2 major transitions. DSC analysis of isolated crude ribosomes aids the assignment of the 2 major peaks observed in whole-cell thermograms to denaturation of ribosomal structures. A comparison of partial and immediate full rescan thermograms of A. pernix whole cells indicates that both major peaks represent irreversible thermal transitions. A DNA peak is also identified in the whole-cell thermogram by comparison with the optical data of isolated pure DNA. DNA melting is shown to be irreversible in dilute solution, whereas it is partially reversible in whole cells, owing at least in part, to restricted volume effects. In contrast to mesophilic organisms, hyperthermophilic A. pernix ribosomes are more thermally stable than DNA, but in both organisms, irreversible changes leading to cell death occur owing to ribosomal denaturation.     

The ultrastructure of the Sertoli cell of the vervet monkey, Chlorocebus aethiops

The ultrastructure of the Sertoli cell of the vervet monkey was studied using both scanning and transmission electron microscopic techniques. SEM micrographs revealed perforated sleeve-like processes which encased mature elongated spermatids which are ready for spermiation. TEM micrographs showed a large Sertoli cell nucleus characterized by many lobes (4–5) and consisting of a homogenous nucleoplasm and a distinctive nucleolus. The nucleus occupies a significant portion of the basal region of the cell. The distribution of chromatin clearly shows high activity of these cells. Lipid droplets and free ribosomes are also found scattered throughout the cytoplasm. Well-developed Golgi apparatus is found in the basal region of the cell. There is phagocytic activity in the Sertoli cells as revealed by the presence of numerous phagosomes. Numerous mitochondria with well-developed tubular cristae are found on the basal side of the nucleus, whereas few mitochondria are located on the apical side of the nucleus. Distinct desmosomes are located between cells. A well-developed smooth endoplasmic reticulum and granular endoplasmic reticulum are frequently found in the cytoplasm of the Sertoli cells. The results of this investigation showed that Sertoli cells of the vervet monkey are almost similar to those of humans and show many similarities with other mammalian species.     

Evaluation of the heat inactivation of Escherichia coli and Lactobacillus plantarum by differential scanning calorimetry

Differential scanning calorimetry (DSC) is used to evaluate the thermal stability and reversibility after heat treatment of transitions associated with various cellular components of Escherichia coli and Lactobacillus plantarum. The reversibility and the change in the thermal stability of individual transitions are evaluated by a second temperature scan after preheating in the DSC to various temperatures between 40 and 130 degrees C. The viability of bacteria after a heat treatment between 55 and 70 degrees C in the DSC is determined by both plate count and calorimetric data. The fractional viability values based on calorimetric and plate count data show a linear relationship. Viability loss and the irreversible change in DSC thermograms of pretreated whole cells are highly correlated between 55 and 70 degrees C. Comparison of DSC scans for isolated ribosomes shows that the thermal stability of E. coli ribosomes is greater than that of L. plantarum ribosomes, consistent with the greater thermal tolerance of E. coli observed from viability loss and DSC scans of whole cells.     

Cellular organisation and differentiation of organelles in pre-meiotic rice anthers

Pre-meiotic cellular organisation of rice anthers has a great significance in pollen formation. We have used a combination of confocal laser and transmission electron microscopy (TEM) to characterise and differentiate organelles in pre-meiotic rice anthers. Along with the characteristic organelles in the cytoplasm the epidermal cells of the pre-meiotic rice anther are coated on their outer surface by a conspicuous bi-lamellate cuticle. Chloroplasts of the endothecium contain immature grana, thylakoids and also starch granules. These plastids clearly contain photosynthetic pigments as shown by autofluorescence in confocal microscope studies. Both confocal and TEM studies reveal clusters of mitochondria in the middle layer. The tapetum contains electron opaque ribosomes, bundles of mitochondria and plastids. The nuclei of the tapetum occupy a large volume of the cytoplasm indicating the onset of mitotic prophase. Intense Rhodamine 123 staining reveals that a major portion of the structurally indistinguishable organelles that were seen throughout the densely ribosomic cytoplasm of sporogenous cells are mitochondria.     

The effect of high hydrostatic pressure on Salmonella thompson and Listeria monocytogenes examined by electron microscopy

Cells of Listeria monocytogenes that had been exposed to pressure contained vacuolar regions in the cytoplasm. Pressure-treated cells of Salmonella thompson contained no vacuoles but had fewer ribosomes than untreated cells and their appearance suggested that some cell lysis had occurred. In both organisms changes in the appearance of the nuclear material were observed.     

Fine structure of the developing oocytes in adultArgas (Persicargas) arboreus (Ixodoidea: Argasidae)

The structure of the developing oocytes in the ovary of unfed and fed femaleArgas (Persicargas) arboreus is described as seen by scanning (SEM) and transmission (TEM) electron microscopy. The unfed female ovary contains small oocytes protruding onto the surface and its epithelium consists of interstitial cells, oogonia and young oocytes. Feeding initiates oocyte growth through the previtellogenic and vitellogenic phases of development. These phases can be observed by SEM in the same ovary.The surface of isolated, growing oocytes is covered by microvilli which closely contact the basal lamina investing the ovarian epithelium and contains a shallow, circular area with cytoplasmic projections and a deep pit, or micropyle, at the epithelium side. In more advanced oocytes the shell is deposited between microvilli and later completely covers the surface.Transmission EM of growing oocytes in the previtellogenic phase reveals nuclear and nucleolar activity in the emission of dense granules passing into the cytoplasm and the formation of surface microvilli. The cell cytoplasm is rich in free ribosomes and polysomes and contains several dictyosomes associated with dense vesicles and mitochondria which undergo morphogenic changes as growth proceeds. Membrane-limited multivesiculate bodies, probably originating from modified mitochondria, dictyosomes and ribosomal aggregates, are also observed. Rough endoplasmic reticulum is in the form of annulate lamellae. During vitellogenesis, proteinaceous yolk bodies are formed by both endogenous and exogenous sources. The former is involved in the formation of multivesicular bodies which become primary yolk bodies, whereas the latter process involves internalization from the haemolymph through micropinocytosis in pits, vesicles and reservoirs. These fuse with the primary yolk bodies forming large yolk spheres. Glycogen and lipid inclusions are found in the cytoplasm between the yolk spheres.     

Scanning and transmission electron microscopy of the tegument of Paranaella luquei Kohn, Baptista-Farias & Cohen, 2000 (Microcotylidae, Monogenea), parasite of a Brazilian catfish, Hypostomus regani

The surface topography and ultrastructure of the tegument of Paranaella luquei Kohn, Baptista-Farias & Cohen, 2000, a microcotylid monogenean parasite from the gills of Hypostomus regani (Ihering, 1905) (Loricariidae) was studied by scanning (SEM) and transmission electron microscopy (TEM). By SEM, it was observed that the tegument presents transversal ridges, forming folds in the ventral and dorsal surfaces and microvillous-like tegumental projections in the anterior and median regions of body. These projections were also observed by TEM. The tegument is made up of a syncytium delimited by apical and basal plasma membranes, containing inclusion bodies and mitochondria, connected to the nucleated region by means of cytoplasmatic processes. The tegumental cells present a well developed nucleus and cytoplasm containing inclusion bodies, similar to those found on the external layer, mitochondria, rough endoplasmatic reticulum and free ribossomes.     

Comparative ultrastructure of Clara cells in neonatal and older cattle

Calf lungs were fixed with glutaraldehyde and examined by scanning (SEM) and transmission (TEM) electron microscopy to compare the ultrastructure of Clara cells in terminal bronchioles of neonatal calves and older cattle. In the neonatal calf, SEM revealed numerous smooth-surfaced Clara cells protruding above a similar number of ciliated cells, whereas in older animals the surface of Clara cells was lobulated. Thin sections examined by TEM revealed numerous cuboidal to columnar Clara cells with indented nuclei and a pale cytoplasm filled with faintly granular glycogen in the neonatal calf. Some cells were characterized by apical dense and/or pale membrane-bound granules or secretory droplets. Many cells had an apical tubular network of cisternae that were partly smooth and partly decorated with ribosomes. Ultrastructural comparison of Clara cells in a 2-day-old calf with those of 14- and 19-day-old, 4- and 5. 5-month-old, and 3.5-year-old cattle revealed a striking reduction in the amount of glycogen per cell after 14 days. The number of cells with apical granules was small at all ages, and the density of the secretory granules varied greatly in different cells. A variable amount of smooth endoplasmic reticulum (SER) was present but was less prominent than cisternae of ribosomal endoplasmic reticulum (RER). In older cattle, the limited amount of SER compared to the RER and secretory granules suggests that bovine Clara cells are more likely to be secretory than detoxifying.     

CONCOMITANT EFFECTS OF INSULIN ON SURFACE MEMBRANE CONFORMATION AND POLYSOME PROFILES OF SERUM-STARVED BALB/C 3T3 FIBROBLASTS

By scanning and transmission electron microscopy we have shown that insulin rapidly reversed changes in surface membrane conformation and polysome profile induced by the transfer of actively growing Balb/c 3T3 fibroblasts from a serum-containing to a serum-free medium. Morphometric analysis of polysome profiles revealed a 94% aggregation of total f ribosomes during logarithmic growth. This figure fell to 78% after 18 h of serum starvation. The number of f ribosomes per unit area of cytoplasm also fell. 1 h of insulin treatment restored aggregation to 92% and increased the number of f ribosomes per unit area of cytoplasm by 22%. Scanning electron microscopy of logarithmically growing cells revealed an abundance of surface microvilli, whereas serum starvation promoted a smooth surface with few microvilli. After 1 h of insulin treatment, microvilli reappeared with a distribution and subcellular organization characteristic of exponential growth. This study shows the combined and rapid effect of insulin on the regulation of polysome formation and the promotion of a specific surface membrane conformation in cultured cells. The observations are consistent with the knowledge that insulin, acting on the surface membrane, can influence such parameters as membrane transport, and the rates of protein and RNA synthesis.     

In vitro deposition of hydroxyapatite on cortical bone collagen stimulated by deformation-induced piezoelectricity

In the present work, we have studied the effect of the piezoelectricity of elastically deformed cortical bone collagen on surface using a biomimetic approach. The mineralization process induced as a consequence of the piezoelectricity effect was evaluated using scanning electron microscopy (SEM), thermally stimulated depolarization current (TSDC), and differential scanning calorimetry (DSC). SEM micrographs showed that mineralization occurred predominantly over the compressed side of bone collagen, due to the effect of piezoelectricity, when the sample was immersed in the simulated body fluid (SBF) in a cell-free system. The TSDC method was used to examine the complex collagen dielectric response. The dielectric spectra of deformed and undeformed collagen samples with different hydration levels were compared and correlated with the mineralization process followed by SEM. The dielectric measurements showed that the mineralization induced significant changes in the dielectric spectra of the deformed sample. DSC and TSDC results demonstrated a reduction of the collagen glass transition as the mineralization process advanced. The combined use of SEM, TSDC, and DSC showed that, even without osteoblasts present, the piezoelectric dipoles produced by deformed collagen can produce the precipitation of hydroxyapatite by electrochemical means, without a catalytic converter as occurs in classical biomimetic deposition.     

Dorsal-ventral differentiation in Simonsiella and other aspects of its morphology and ultrastructure

The morphology and ultrastructure of the aerobic, Gram-negative multicellular-filamentous bacteria of the genus Simonsiella were investigated by scanning and transmission electron microscopy. The flat, ribbon-shaped, multicellular filaments show dorsal-ventral differentiation with respect to their orientations to solid substrata. The dorsal surface, orientated away from the substrate, is convex and possesses an unstructured capsule. The ventral surface, on which the organisms adhere and glide, is concave and has an extracellular layer with fibrils extending at right angles from the cell wall. The cytoplasm in the ventral region contains a proliferation of intracytoplasmic membranes and few ribosomes in comparison to the cytoplasm in other parts of the cell. Centripetal cell wall formation is asymmetrical and commences preferentially in the ventral region. Quantitative differences in morphology and cytology exist among selected Simonsiella strains. Functional aspects of this dorsalventral differentiation are discussed with respect to the colonization and adherence of Simonsiella to mucosal squamous epithelial cells in its ecological habitat, the oral cavities of warm-blooded vertebrates.List of Abbreviations SEM scanning electron microscope - TEM transmission electron microscope     

Lethal effect of chitosan‐Ag (I) films on Staphylococcus aureus as evaluated by electron microscopy

Aim: This article investigated the lethal effect and morphological changes on Staphylococcus aureus strains ATCC 25923 and ATCC 6538P produced by chitosan‐Ag (I) films as observed by electron microscopy. Methods and Results: The antimicrobial activity of films against staphylococci was determined using the broth dilution method and agar diffusion test. Killing curves, transmission and scanning electron microscopy (TEM and SEM) techniques were employed to evaluate the bacterial death and morphological changes in bacterial cells after exposure to chitosan‐Ag (I) films. Films affected the cell structure of Staph. aureus, causing elongation of cells, disaggregation of grape‐like cluster, contraction of bacterial cytoplasm, thickening of cell wall, increase in cell wall roughness, cell disruption with loss of intracellular material, filamentation and bacteriolysis, as seen in the micrographs following 3, 6, 12 and 16 h of incubation. Conclusions: Obtained images clearly show that chitosan‐Ag (I) films have a notable antistaphylococcal activity. Significance and Impact of the Study: Information from this study can be employed in guiding future strategies to improve the design of materials for the food industry packaging.     

An electron microscopic examination of the gastrointestinal epithelium in Dover sole, Solea solea (L.)

The gastrointestinal tracts of adult and juvenile Dover sole, Solea solea (L.), were examined using scanning (SEM) and transmission electron microscopy (TEM). SEM showed little differentiation of the internal morphology of the gastrointestinal tract in adult fish, with longitudinally arranged mucosal folds present in all gut regions. Mucosal folds had a similar arrangement to that in the goldfish. Goblet cells were identified in the mucosal epithelium in all regions of the gut while microscopic ducts/pores of possible pancreatic origin were observed in the foregut region. SEM of juvenile gut samples showed a similar arrangement of longitudinal mucosal folds to that found in the adult fish. There was no visible evidence of goblet cells or secretory ducts/pores in any region of the juvenile gastrointestinal tract.
In TEM it was observed that apical microvilli were a feature of epithelial cells from all regions of the gastrointestinal tract in adult and juvenile Dover sole. Cells from the distal regions of the adult and juvenile gut showed invaginations and vacuolation of the apical cytoplasm. A high degree of vacuolation in cells from the juvenile hindgut-rectum indicated the possible occurrence of intracellular digestion of absorbed nutrients in this gut region.     

Whole-cell-mount cytochemistry by the colloidal gold labeling method. Combined transmission and scanning electron microscopic study of ConA binding sites in mouse macrophages

Binding, redistribution, and endocytosis of colloidal gold (CG)-labeled concanavalin A (ConA) were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Mouse peritoneal macrophages were cultured on Formvar-coated platinum grids. Either fixed or unfixed cells were labeled by the indirect ConA-CG labeling method. Specimens were critical-point-dried and observed by TEM and SEM in the same region. Surface-bound ConA-CG was easily seen by SEM. Stereomicroscopic observation by TEM clearly showed the three-dimensional distribution of ConA on the cell surface as well as in the cytoplasmic vesicles and vacuoles. In the prefixed cells, CG was distributed randomly on the cell surface. When unfixed cells were labeled at 0 degree C, a similar binding pattern was observed, although the density of bound CG was decreased. When cells labeled with ConA-CG at 0 degree C were further incubated at 37 degrees C, redistribution and endocytosis of the label were seen. Endocytosed CG in the cytoplasmic vesicles and vacuoles was clearly seen by TEM. In addition, three-dimensional location and relationship with other organelles were easily observed. Combined TEM and SEM observation of CG-labeled whole-cell-mount specimens is a useful method to study the dynamics of cell-bound ligands.     

Characterization of methylglyoxal-modified human IgG by physicochemical methods

Human IgG is a defence protein and quite reactive to dicarbonyls. In this study, methylglyoxal-induced modification of IgG was examined by various biochemical and biophysical methods. The methylglyoxal-induced changes in IgG were monitored by UV-visible and fluorescence spectroscopy, Fourier transform infrared spectroscopy, 1-anilinonaphthalene-8-sulfonic acid (ANS), and thermal denaturation studies. Aggregate formation was studied by Thioflavin T (ThT), Congo red (CR) and scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Spectroscopic studies suggested gross changes in MGO-modified IgG. Fluorogenic AGEs appeared during modification and the MGO-modified IgG gained thermostability. The reaction produced oxidative stress in the medium because carbonyl content increased manifold and sulfhydryl groups decreased. Enhanced binding of the MGO-modified IgG by Congo red and Thioflavin T suggests crosslinking and aggregation. This was supported by SEM and TEM results.     

Structural stability of ribosomes subjected to RNase treatment evidenced by dielectric spectroscopy and differential scanning microcalorimetry

Previous studies from our laboratory demonstrated the existence of at least two levels of structural complexity in E. coli 70S ribosomes. Ribosomal RNA seems to be principally involved in the overall stability of these structures. In this paper we present an investigation of ribosomes subjected to treatment with RNase. The study is based on both differential scanning microcalorimetry and dielectric spectroscopy. In the thermograms obtained on treated ribosomes only the low temperature peak of the two typical denaturation events observed in native ribosomes, is promptly eliminated by the enzyme treatment. Dielectric spectroscopy measurements carried out on the same samples indicate an alteration of the dielectric behavior previously shown to consist of two subsequent relaxation processes. In fact, only the low frequency relaxation is affected by the treatment. The second one, observed at higher frequency, remains unaltered. The same effect on the dielectric parameters is observed if the ribosome particles are heated and then cooled prior to measurement. These results are consistent with the idea that two different structures are present within the ribosome. One is very stable and withstands both temperature and RNase treatment while the second is promptly abolished by both treatments. Data presented here strongly suggest that the RNA domains exposed to the solvent play a fundamental role in the stability of the 3-D structure of the ribosome particle.     

The cytoplasmic organization of hyphal tip cells in the fungusAllomyces macrogynus

Summary Light and transmission electron microscopy were used to examine hyphal tip cells of the fungusAllomyces macrogynus (Chytridiomycetes). A well defined apical body, i.e., Spitzenkörper, was observed at the extreme apex of hyphal cells. This distinctive, spherical cytoplasmic region consisted of a granular matrix devoid of ribosomes and most organelles. To our knowledge this is the first report describing such a structure in hyphae of an aseptate fungus. Vesicles (45–65 nm diameter) were concentrated in the peripheral cytoplasm of the apex, while relatively few were observed within the Spitzenkörper. Filasomes, spherical patches of dense fibrillar material containing a microvesicle core, were abundant in the apical regions near the plasma membrane. Microtubules traversed the Spitzenkörper at various angles and were in close association with the plasma membrane. Microfilaments were observed as individual elements in the cytoplasm or were organized into bundles. Individual microfilaments were frequently in close association with the plasma membrane, vesicles and microtubules. In the immediate subapical region mitochondria, multivesicular bodies, microbodies, Golgi equivalents and nuclei were abundant.Abbreviations CW cell wall - F filasome - M mitochondria - N nucleus - PM plasma membrane - TEM transmission electron microscopy     

Membrane blebbing is associated with Ca2+-activated hyperpolarizations induced by serum and alpha 2-macroglobulin

We have reported previously that serum and alpha 2-macroglobulin (alpha 2M) induce Ca2+-activated hyperpolarizations in the membrane potential of a clonal rat osteosarcoma cell line (ROS 17/2) (Dixon and Aubin, J. Cell, Physiol., 132:215-225, 1987). In this report, we describe morphological changes that accompany these hyperpolarizations. Both cell surface blebbing (zeiosis) and transient hyperpolarizations were induced by application of 10% fetal bovine serum (FBS) or alpha 2M; neither was induced by serum-free medium, a suspension of latex beads, or purified bovine serum albumin. Following a brief application of FBS or alpha 2M at time 0, electrical activity typically occurred between 7-40 s and was always followed by blebbing activity that began at 30 s and persisted for 3-5 min. In contrast, continuous exposure to FBS resulted in the persistence of both blebbing activity and transient hyperpolarizations for periods of at least several hours. Scanning electron microscopy (SEM) revealed that the blebs appeared concomitantly with the disappearance of microvilli and the appearance of surface pits that measured 100-300 nm in diameter. Coated pits and vesicles, similar in size to the pits observed by SEM, were observed using transmission electron microscopy (TEM). By TEM, blebs were found to contain few organelles other than centrally located free ribosomes. Fluorescence microscopy of nitrobenzooxadizole-phallacidin-labeled cells indicated that blebs contained filamentous actin and that microfilament bundles remained primarily on the substratum side of blebbed cells. We propose that blebbing results from a dynamic local reorganization of microfilaments initiated by ligand-induced transient increases in intracellular Ca2+.     

Electron-microscopical study of the choroid plexus and epiplexus cells in cats following a cisternal injection of crotoxin complex

The choroid plexus and its associated epiplexus cells in the fourth ventricle in cats were studied with scanning and transmission electron microscopy (SEM, TEM) following a cisternal injection of crotoxin complex (phospholipase A2). In SEM, the epiplexus cells of the control animals were predominantly stellate with long radiating processes. At 2 h after the administration of crotoxin complex, these radiating processes flattened out forming sheet-like membranes covering the ventricular surface of the choroid epithelial cells. The membranous coverings remained extended in 5-hour-survival cats. Numerous blebs of different sizes were observed in areas that were not covered by the cytoplasmic membrane in 5-hour animals. Some of the blebs appeared to have ruptured. In TEM, the microvilli of the choroid epithelial cells in crotoxin complex-treated rats were dilated. The luminal surface of the epithelial cells showed eruption of blebs filled with amorphous materials. Pinocytotic vesicles increased in number in the apical cytoplasm. The lumen of the ventricle often contained portions of cytoplasm believed to be derived from the extrusion of the blebs. These appeared to be engulfed by the overlying epiplexus cells. It was concluded that the injected crotoxin complex stimulated both the secretory as well as pinocytotic activity of the choroid epithelial cells. The phagocytosis of the secretory products from the epithelial cells by epiplexus cells suggests a close functional relationship between the two cell types.