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PubMed1999

Direct probing of the surface ultrastructure and molecular interactions of dormant and germinating spores of Phanerochaete chrysosporium.

Atomic force microscopy (AFM) has been used to probe, under physiological conditions, the surface ultrastructure and molecular interactions of spores of the filamentous fungus Phanerochaete chrysosporium. High-resolution images revealed that the surface of dormant spores was uniformly covered with rodlets having a periodicity of 10 +/- 1 nm, which is in agreement with earlier freeze-etching measurements. In contrast, germinating spores had a very smooth surface partially covered with rough granular structures. Force-distance curve measurements demonstrated that the changes in spore surface ultrastructure during germination are correlated with profound modifications of molecular interactions: while dormant spores showed no adhesion with the AFM probe, germinating spores exhibited strong adhesion forces, of 9 +/- 2 nN magnitude. These forces are attributed to polysaccharide binding and suggested to be responsible for spore aggregation. This study represents the first direct characterization of the surface ultrastructure and molecular interactions of living fungal spores at the nanometer scale and offers new prospects for mapping microbial cell surface properties under native conditions.

Microscopy, Atomic Force↗
PubMed1965

Ultrastructure and Ribosomes of Mycoplasma gallisepticum.

Maniloff, Jack (Yale University, New Haven, Conn.), Harold J. Morowitz, and Russell J. Barrnett. Ultrastructure and ribosomes of Mycoplasma gallisepticum. J. Bacteriol. 90:193-204. 1965.-The ultrastructure of Mycoplasma gallisepticum strain A5969 has been studied by electron microscopy (thin-section and negative staining), ultracentrifugation, and chemical analysis. The list of ultrastructure is: membrane, nuclear material, ribosomes, ribosomal structures, infra-bleb region, and blebs. The nuclear material, containing the cell's deoxyribonucleic acid, appears as an unbounded region containing 30-A fibrils. The ribosomes have a diameter of about 140 A, a ribonucleic acid-protein ratio of 0.68, and an uncorrected sedimentation coefficient of 70.2S. The 70.2S particle can be broken into 49.3S and 32.4S particles. Ribosomal arrays were found filling the intracytoplasmic space between the nuclear material and the membrane. Under certain conditions, these arrays formed cylindrical arrangements of ribosomes. The infra-bleb region is composed of a granular material, although little internal structure could be found. The bleb was highly structured.

Journal Article↗
PubMed1966

Utility of sodium hypochlorite for ultrastructure study of bacterial spore integuments.

Rode, L. J. (The University of Texas, Austin), and M. Glenn Williams. Utility of sodium hypochlorite for ultrastructure study of bacterial spore integuments. J. Bacteriol. 92:1772-1778. 1966.-Spores of Bacillus megaterium are partially dissolved by sodium hypochlorite. Spore integuments become visible during the dissolution, and ultrastructural features may be detected. Three distinct integument types are described for B. megaterium QM B1551 with the use of this technique. Since a variety of microbial cells are affected by sodium hypochlorite, its use may be applicable to ultrastructure study of cells other than bacterial spores.

Bacillus megaterium↗
PubMed1969

Purification, ultrastructure, and composition of axial filaments from Leptospira.

The ultrastructure of three strains of water Leptospira was studied by negative staining, thin sectioning, and freeze-etching. The cells possessed a triple-layered sheath which covered two independent axial filaments, one inserted subterminally in each end of the cell. The protoplasmic cylinder was surrounded by a triple-layered cell wall and possessed ribosomes, lamellar structures, and a typical procaryotic nuclear region. The axial filament was comprised of several component structures. An axial fibril, with a diameter of 20 to 25 nm, consisted of a solid inner core (13 to 16 nm in diameter) surrounded by a coat. A terminal knob (40 to 70 nm in length) was connected to a series of disc insertion structures at the terminal end of the axial fibril. The axial fibril was surrounded by a helical outer coat (35 to 60 nm in diameter) which was composed of a continuously coiled fiber, 3 to 4 nm in diameter, embedded in an electron-dense material. A procedure for the purification of the axial fibrils was presented and their ultrastructural, physical, and chemical properties were determined. Similarities in ultrastructural, physical, and chemical properties were noted between the axial fibrils and bacterial flagella. A schematic model of the leptospiral axial filament is presented, and a mechanism is proposed for its function as a locomotor organelle.

Amino Acids↗
PubMed1989

Surface properties and ultrastructure of Edwardsiella species.

Twenty-two strains of Edwardsiella representing the three currently recognized species were evaluated for surface characteristics and ultrastructural morphology. All isolates tested possessed a high surface cell charge as detected by DEAE-cellulose chromatography; surface hydrophobicity was variable and strain but not species dependent. Two major types of adhesins detected by hemagglutination assays were identified: one was inhibited by D-mannose (mannose-sensitive hemagglutinin) and found on all three species, and a second was not inhibited by D-mannose (mannose-resistant hemagglutinin) and was principally associated with Edwardsiella tarda. The results of physiologic and ultrastructural studies suggest that both hemagglutinins are afimbrial adhesin proteins. The results of these studies suggest that there are a number of distinct surface and ultrastructural properties associated with each of the three Edwardsiella species.

Chromatography, DEAE-Cellulose↗
PubMed2004

Clinical and ultrastructural findings in mare's tail lines of the corneal epithelium.

AIMS: Mare's tail lines are uncommon, grey, parallel, tapering epithelial lines, which may give rise to discomfort or to visual symptoms. The authors report the clinical and ultrastructural findings in two patients METHODS: Therapeutic debridement was performed in two patients suffering from mare's tail disorder. The loosely attached epithelium was removed and fixed in glutaraldehyde containing cuprolinic blue and processed in a standard fashion for electron microscopy. RESULTS: Ultrastructural studies showed an avascular, collagenous pannus extending under the whole of the excised epithelium in each case and separated from it by a thickened basal lamina like zone. The basal laminar material extended into the epithelium in folds, in keeping with the clinical features. Interesting ultrastructural features included: (1) a lack of hemidesmosomes in relation to the basal laminar material; (2) trapping of degenerate keratocytes within the invaginating basal laminar folds; (3) an unusual regularity and fine dimensions of the collagen fibrils and proteoglycans making up the subepithelial pannus. The basal laminar material contained proteoglycans and small fibres similar in appearance to long spacing collagen. CONCLUSIONS: Mare's tail lines are caused by basal laminar material, assumed to be chiefly of epithelial origin, which invaginates the corneal epithelium. The presence of occasional keratocytes within the invaginating folds suggests that there may be a stromal contribution to the disorder. The visibility of mare's tail lines in the focal beam of the slit lamp is likely to be related to the thickness and light scattering properties of the invaginations and of the subepithelial deposits. Tractional forces, imposed by lid action, could explain their horizontal disposition.

Aged↗
PubMed1995

Histological and ultrastructural investigation of retinal microaneurysm development in diabetic patients.

BACKGROUND: Although microaneurysms are a clinicopathological hallmark of diabetic retinopathy, there have been few ultrastructural studies of these important lesions. As a result, knowledge of the mechanisms involved in the pathogenesis of microaneurysms remains fragmentary. This study provides histological and ultrastructural evidence of various stages in microaneurysm formation within the retinal vasculature. METHODS: The eyes of three type II diabetic patients, obtained within 24 hours of death, were studied by the trypsin digest technique. Eyes from two further type II diabetics were fixed in 2.5% glutaraldehyde within 12 hours of death and processed for electron microscopy. RESULTS: In the trypsin digest preparations, small saccular and fusiform microaneurysms were observed in the peripheral retinal. In the central retina, the microaneurysms ranged in morphology from thin walled, cellular forms to dense, acellular, hyalinised forms. Ultrastructurally, four distinct groups of microaneurysm were observed. Type I showed an extensive accumulation of polymorphonuclear cells into the lumen. The endothelium remained intact, although pericytes were invariably absent. Type II microaneurysms were typified by large numbers of red blood cells (RBCs) in the lumen. Endothelial cells and pericytes were completely absent. The type III microaneurysm was also non-perfused and contained aggregates of irregularly shaped RBC profiles and RBC breakdown products. Recanalisation by new vessels into the occluded lumen was observed in one microaneurysm. Type IV microaneurysms were almost or completely sclerosed, with extensive fibrosis and lipid infiltration into the lumen and basement membrane wall. CONCLUSION: This investigation describes several distinctive stages in the formation of microaneurysms during diabetic retinopathy. With reference to the pathogenesis of retinal microaneurysms, the interaction of various cell types is discussed and the significance of vascular cell death and localised hypertensive events highlighted.

Aneurysm↗
PubMedDate not supplied

Extending unbiased stereology of brain ultrastructure to three-dimensional volumes.

OBJECTIVE: Analysis of brain ultrastructure is needed to reveal how neurons communicate with one another via synapses and how disease processes alter this communication. In the past, such analyses have usually been based on single or paired sections obtained by electron microscopy. Reconstruction from multiple serial sections provides a much needed, richer representation of the three-dimensional organization of the brain. This paper introduces a new reconstruction system and new methods for analyzing in three dimensions the location and ultrastructure of neuronal components, such as synapses, which are distributed non-randomly throughout the brain. DESIGN AND MEASUREMENTS: Volumes are reconstructed by defining transformations that align the entire area of adjacent sections. Whole-field alignment requires rotation, translation, skew, scaling, and second-order nonlinear deformations. Such transformations are implemented by a linear combination of bivariate polynomials. Computer software for generating transformations based on user input is described. Stereological techniques for assessing structural distributions in reconstructed volumes are the unbiased bricking, disector, unbiased ratio, and per-length counting techniques. A new general method, the fractional counter, is also described. This unbiased technique relies on the counting of fractions of objects contained in a test volume. A volume of brain tissue from stratum radiatum of hippocampal area CA1 is reconstructed and analyzed for synaptic density to demonstrate and compare the techniques. RESULTS AND CONCLUSIONS: Reconstruction makes practicable volume-oriented analysis of ultrastructure using such techniques as the unbiased bricking and fractional counter methods. These analysis methods are less sensitive to the section-to-section variations in counts and section thickness, factors that contribute to the inaccuracy of other stereological methods. In addition, volume reconstruction facilitates visualization and modeling of structures and analysis of three-dimensional relationships such as synaptic connectivity.

Algorithms↗
PubMed2004

Sertoli cell nodules in the undescended testis: a histochemical, immunohistochemical, and ultrastructural study of hyaline deposits.

AIMS: To document the morphology, immunohistochemical staining properties, and ultrastructural features of hyaline material in Sertoli cell nodules of undescended testis and contrast them with those of sex cord tumour with annular tubules (SCTAT), which is histologically similar. To highlight the need to distinguish these nodules from other Sertoli cell hyperplasias, such as intratubular Sertoli cell proliferations, which occur in specific clinical contexts. MATERIALS/METHODS: A retrospective study of 46 orchidectomy specimens from cryptorchid testes, 27 of which contained Sertoli cell nodules. Special histochemical stains, immunohistochemical stains for type IV collagen and fibronectin, and ultrastructural examination of the hyaline material were performed using tissue from paraffin wax embedded tissue blocks. RESULTS: The hyaline deposits in SCTAT and Sertoli cell nodules had similar staining patterns-periodic acid Schiff (PAS) and PAS-diastase positivity with variable staining of Martius scarlet blue and Masson trichrome. Type IV collagen immunoreactivity was seen in hyaline areas, although fibronectin was negative. Electron microscopy of hyaline areas confirmed a compact matrix identical to components of the basement membrane in the adjacent seminiferous tubules. CONCLUSION: This study describes an unusual form of Sertoli cell proliferation in undescended testes, which must be distinguished from Sertoli cell tumours and other forms of proliferation. In addition, the hyaline material within Sertoli cell nodules in the cryptorchid testis is histochemically, immunohistochemically, and ultrastructurally consistent with both matrix and fibrous components of seminiferous tubule basement membranes. Increased production of basement membrane material, with subsequent invagination into tubules, is the most likely origin of this material.

Adolescent↗
PubMed1995

In situ end-labelling, light microscopic assessment and ultrastructure of apoptosis in lung carcinoma.

AIMS: To compare in situ end-labelling (ISEL) of apoptosis in lung carcinoma with quantitative and semiquantitative light microscopic assessment and ultrastructural observations. METHODS: ISEL of apoptosis was evaluated in 42 lung carcinomas (24 squamous cell carcinomas, 12 adenocarcinomas and six small cell carcinomas). Results were correlated semiquantitatively with the extent of apoptosis in haematoxylin and eosin stained sections, with apoptotic indices and with ultrastructural observations (nine cases). RESULTS: In each tumour type the extent of apoptosis identified by ISEL correlated with that observed on light and electron microscopy. Tumour cells undergoing apoptosis showed either uniform nuclear staining with a surrounding "halo" or peripheral nuclear membrane staining. The latter pattern was more prominent in small cell carcinoma and correlated ultrastructurally with early apoptosis. A variable proportion of apoptotic cells and apoptotic bodies were unlabelled. Necrotic tumour cells were weakly stained but were distinguishable from apoptotic cells. CONCLUSIONS: ISEL, if used in conjunction with standard methods for investigating apoptosis, is a useful adjunct to the investigation of apoptosis in human tumour tissue.

Adenocarcinoma↗
PubMed1995

Early ultrastructural changes of antral mucosa with aspirin in the absence of Helicobacter pylori.

AIMS: To describe the ultrastructural changes that occur in human antral mucosa following direct application of aspirin in volunteers without Helicobacter pylori infection. METHODS: Ten healthy male volunteers without H pylori infection underwent three consecutive endoscopies (at zero, one and five hours). At the first endoscopy, two biopsy specimens were obtained (one for histology and the other for electron microscopy (EM)). At subsequent endoscopies, a single biopsy specimen was obtained for EM. A 50 ml solution of aspirin (concentration 3 mg/ml) was applied to the antral mucosa at the first endoscopy in five subjects; the other five subjects received 50 ml distilled water (placebo). RESULTS: The ultrastructural appearance of the first biopsy specimen in all subjects and subsequent biopsy specimens in the placebo treated subjects was normal. The aspirin treated group had evidence of intercellular oedema, widening of capillary fenestrae, rupturing of apical membranes, and dilatation of endoplasmic reticulum and mitochondria after one hour; these changes were more marked at five hours. Tight junctions were maintained. CONCLUSION: This is the first study to describe the early ultrastructural changes in antral mucosa induced by aspirin in subjects without H pylori infection.

Adult↗
PubMed1991

Ultrastructural aspects of infection with Treponema pallidum subspecies pertenue (Pariaman strain).

OBJECTIVE: To study ultrastructural aspects of infection with Treponema pertenue (Pariaman strain), originating from West Sumatra, Indonesia. MATERIALS AND METHODS: Biopsy material originating from skin lesions in ten young children suffering from early infectious yaws in Indonesia, and rabbit testicular tissue inoculated with T pertenue. Human skin as well as rabbit testicular tissue was examined by means of conventional electron microscopy. RESULTS: In human skin, treponemes were found in interepidermal spaces in 5 out of 10 specimens. In two of five positive specimens, treponemes were also seen in the dermis. In one out of five specimens from rabbit testicular tissue a profusion of treponemes was found lying in the interstitial myxomatous tissue. Microorganisms showed no adhesion to fibroblasts. CONCLUSION: This ultrastructural study of T pertenue demonstrated the scarcity and focal distribution of treponemes in tissue and did not reveal any morphological differences from the Gauthier strain of T pertenue. No differences from the ultrastructure of T pallidum were observed either.

Animals↗
PubMed1993

Muscle ultrastructure after strength training with placebo or anabolic steroid.

The influence of strength training on muscle fiber area, capillary ultrastructure, and capillary supply was studied in experienced male bodybuilders taking either anabolic steroids or a placebo. During 8 weeks, 13 subjects received a weekly injection of either placebo or nandrolone decanoate (starting dose 200 mg, followed by 100 mg weekly), in a double-blind, counterbalanced design. Before and after 8 weeks a needle biopsy was taken from the vastus lateralis muscle for studying morphometric characteristics of fibers and ultrastructure and morphometric characteristics of capillaries. The fiber cross-sectional area increased significantly only after steroid treatment. Capillary ultrastructure and capillary supply were not changed after the training period. In none of the measured parameters was any difference found between placebo and steroid treatment.

Adult↗
PubMed1984

Fetal canine cardiac Purkinje fibers: electrophysiology and ultrastructure.

We studied the ontogenesis of the transmembrane action potential and the ultrastructure of fetal canine Purkinje fibers. Fetal hearts were obtained from fetuses just after implantation to end gestation. Using standard microelectrode recording techniques, we found that action potential characteristics varied linearly over this period of development. Maximum diastolic potential (MDP) ranged from -65 to -85 mV; action potential amplitude (AMP) varied from 100 to 120 mV; maximum upstroke velocity (Vmax) increased from 200 to 550 V/s. Action potential duration measured to 50% repolarization (APD50) increased from 15 to 156 ms while duration measured at full repolarization (APD100) similarly increased from 75 to 236 ms. The relationship between external potassium concentration and membrane potential was equivalent across all stages of fetal development. Tetrodotoxin (TTX, 7.7 X 10(-7) to 1.6 X 10(-5) M) caused concentration-dependent decreases in AMP, Vmax, and APD50. Verapamil (1 X 10(-7) to 1 X 10(-5) M) decreased Vmax and APD50 in a concentration-dependent manner. The effects of both TTX and verapamil were statistically equivalent across all stages of fetal development. Ultrastructural studies of fetal Purkinje fibers showed that myocytes at the earliest stages of development (Purkinje fibers were not visually distinct at this time) were arranged as a tightly packed mosaic with a rounded shape, with a large amount of glycogen, small sparse mitochondria, and relatively large nuclei. Mitotic cells were observed frequently. Purkinje fibers when first identified grossly had fewer myofilaments than working myocardial cells and sarcomeres without M lines. By late gestation, intercalated disks appeared with an increase in surface areas; desmosomes occurred more frequently. Myofilaments are organized around Z bands into rudimentary sarcomeres that still lack M lines. These data indicate that, although the fetal canine Purkinje fiber undergoes marked developmental changes in ultrastructure, cellular electrophysiological changes are more subtle. The action potential has a qualitative appearance similar to those of the neonatal or adult fiber. At no time during fetal development could we find slow-response action potentials.

Action Potentials↗
PubMed1975

Effects of mannitol on cardiac ultrastructure and microcirculation following anoxia.

Electron microscopic and microcirculatory effects of hyperosmolal mannitol were evaluated in the isolated perfused isovolumic rat heart. Specimens for ultrastructural examination were obtained in 26 experiemnts after 15 min of sequential aerobic, anoxic, and reoxygenated perfusion using an isosmolal perfusate of Krebs-Ringer-Henseleit bicarbonate buffer (KRB) (osmolality equals 290 mosmol/kg) vs. a hyperosmolal solution of KRB + mannitol (equals 350 mosmol/kg). No significant changes were noted during aerobic perfusion. Anoxic hearts perfused with isosmolal KRB demonstrated the most severe ultrastructural alterations including: mitochondrial swelling with disruption of cristae, myofibrillar fusion and contraction bands, and subsarcolemmal edema and vacuolization. These subcellular changes were not only partially reversed by oxygenated isosmolal perfusion but were significantly reversed during both the anoxic and reoxygenation perfusion periods with mannitol added. Following silicone rubber injection of the microcirculation, only focal capillary endothelial cell swelling was noted, and no difference in arteriolar or capillary filling was observed with either perfusate. Thus, mannitol significantly reversed the postanoxic ultrastructural changes consistently observed in the absence of increased osmolality. No gross effect on vascular patency could be demonstrated.

Animals↗
PubMed2004

Surfactant protein D influences surfactant ultrastructure and uptake by alveolar type II cells.

Surfactant protein D (SP-D) is a member of the collectin family of the innate host defense proteins. In the lung, SP-D is expressed primarily by type II cells. Gene-targeted SP-D-deficient [SP-D(-/-)] mice have three- to fivefold higher surfactant lipid pool sizes. However, surfactant synthesis and secretion by type II cells and catabolism by alveolar macrophages are normal in SP-D(-/-) mice. Therefore, we hypothesized that SP-D might regulate surfactant homeostasis by influencing surfactant structure, thereby altering its uptake by type II cells. Large (LA) and small aggregate (SA) surfactant were isolated from bronchoalveolar lavage fluid (BALF) from SP-D(-/-), wild-type [SP-D(+/+)], and transgenic mice in which SP-D was expressed under conditional control of doxycycline in alveolar type II cells. Uptake of both LA and SA isolated from SP-D(-/-) mice by normal type II cells was decreased. Abnormally dense lipid forms were observed by electron microscopy of LA from SP-D(-/-) mice. SA from SP-D(-/-) mice consisted of atypical multilamellated small vesicles. Abnormalities in surfactant uptake by type II cells and in surfactant ultrastructure were corrected by conditional expression of SP-D in vivo. Preincubation of BALF from SP-D(-/-) mice with SP-D changed surfactant ultrastructure to be similar to that of SP-D(+/+) mice in vitro. The rapid changes in surfactant structure, increased uptake by type II cells, and decreased pool sizes normally occurring in the postnatal period were not seen in SP-D(-/-) mice. SP-D regulates uptake and catabolism by type II cells and influences the ultrastructure of surfactant in the alveolus.

Animals↗
PubMed1984

Quantitative ultrastructure and functional correlates in proximal tubule of Ambystoma and Necturus.

The functional properties of the proximal tubule in the salamander Ambystoma tigrinum have been well characterized but its ultrastructure has not been examined. We therefore analyzed the qualitative and quantitative ultrastructure of the proximal tubule in this species as a basis for structure-function correlations. In addition, a comparative study between species was performed between Ambystoma and Necturus. In Ambystoma the basal cell membrane but not the lateral cell membrane has a highly elaborate organization and is greatly amplified at the basal cell surface. Therefore, the bulk of the basolateral membrane does not face the lateral intercellular space but faces a basal extracellular labyrinth immediately adjacent to the peritubular space. We suggest that this intraepithelial compartment may serve as a site for solute-solvent interactions. The morphometric comparative analysis provides quantitative estimates of tubule dimensions, volume of cells and extracellular channels, areas of luminal, lateral, and basal cell membranes as well as averaged dimensions of the lateral intercellular spaces. Structure-function correlations show that when certain functional parameters are normalized on the basis of ultrastructural rather than epithelial dimensions the interspecies variability decreases.

Ambystoma↗
PubMed1987

Isolated perfused Ambystoma proximal tubule: hydrodynamics modulates ultrastructure.

A method using a pressure-sensing servo-pipette is described for measuring downstream transepithelial pressure within isolated renal tubules perfused at flow rates designed to keep luminal solution composition constant. The hydrodynamics of in vitro microperfusion of isolated proximal tubules of Ambystoma tigrinum was varied and different states of transepithelial hydrostatic pressure difference, axial tubule flow, and transepithelial transport were correlated with epithelial ultrastructure. Tubules analyzed by ultrastructural morphometry were as follows: unperfused with and without ouabain, perfused single-end cannulated with and without ouabain, and perfused double-end cannulated tubules incubated in substrate Ringer. The results indicate that proximal tubule fine structure is well preserved for more than 3 h in unperfused and perfused tubules. Small transepithelial hydrostatic pressure gradients (less than 162 Pa) increase tubule diameters and decrease cell height without changing volumes of the cells, lateral intercellular spaces (LIS), or the basal extracellular labyrinth (BEL). Pressure gradients of 271 Pa have no further effect on tubule diameters or cell height, but significantly reduce volumes of LIS and BEL. Transport inhibition and axial flow changes have minor structural effects. This study demonstrates a close dependence of tubule ultrastructure on hydrodynamic conditions and provides guidelines for optimizing the latter during perfusion of isolated renal tubules.

Ambystoma↗