Fibrillar Layer Research Articles

Pathobiology of articular cartilage in trapeziometacarpal osteoarthritis. II. Surface ultrastructure by scanning electron microscopy

Regional variation in the surface ultrastructure of hyaline cartilage in the osteoarthritic trapeziometacarpal joint was studied by means of scanning electron microscopy. Articular surface degeneration on intact osteochondral specimens was analyzed and correlated with known patterns of gross, histologic, and biochemical disease. The earliest changes were observed in the tangential surface lamina and occurred in areas of chondromalacia in the palmar contact area of the joint. Progressive disruption of this superficial fibrillar layer of the articular cartilage was followed by disorganization of the underlying chondrocytes and matrix. In palmar regions with more advanced chondromalacia and loss of eosinophilic staining, free cellular elements and craters 20–30 μm in diameter, resembling empty chondrocyte lacunae, appeared in conjunction with the absence of any tangential surface meshwork. Eburnated areas demonstrated no fibrillar or cellular elements. Disruption of the protective surface lamina of trapeziometacarpal articular cartilage occurs in a geographic pattern corresponding to joint contact areas and regions of selective biochemical decomposition of proteoglycan matrix. These observations support an interdependent relationship between mechanically induced abrasive surface wear and biochemical matrix degradation in the production of the cartilage lesion of trapeziometacarpal osteoarthritis.

Ultrastructural studies of spore walls of Acaulospora morrowiae and A. scrobiculata

Morphological and ultrastructural studies were used to determine spore wall structure in two Acaulospora species: A. morrowiae and A. scrobiculata. The spore wall of A. morrowiae is divided in three zones (A, B, C), each of which in broken spores separate into layers. Many features, such as a series of regularly distributed fibrillar layers of arched-appearing fibrils were clearly distinguishable. In A. scrobiculata, two zones were distinct in the wall: an outer zone, ornamented with deep round-bottomed depressions which extended into several of the arched-like layers, and an inner zone formed by three distinct layers. The first layer of the inner zone had a radial arrangement of fibrils; the two other layers were formed apparently by amorphous material. Arched and radial fibrils have been found in spores of other Glomales but not in those of other groups of fungi. Spores of Acaulospora are similar to those of certain other glomalean fungi. These and other data indicate the uniqueness of spore walls in the Glomales.

The existence of a dispensable fibrillar layer on the wall surface of mycelial but not yeast cells of Aureobasidium pullulans

The existence of a dispensable fibrillar layer on the wall surface of mycelial but not yeast cells of Aureobasidium pullulans

Observations On the Cuticle Ultrastructure in the Hoplolaiminae (Nemata: Hoplolaimidae)

The fine structure of the cuticle of Aorolaimus macbethi, Aphasmatylenchus straturatus, A. variabilis, Helicotylenchus dihystera, H. multicinctus, Hoplolaimus pararobustus, H. seinhorsti and Pararotylenchus hopperi is described. Six layers are identified in Aphasmatylenchus, Helicotylenchus and Pararotylenchus species vs seven in Hoplolaimus and Aorolaimus species. The ultrastructure of the five outer layers of the cuticle is identical in all species and consists of an external cortex, an internal cortex, a granular or fibrillar layer with electron dense ovoid structures, a striated layer and an electron dense fibrillar layer; the basal zone of the cuticle consists of a thin electron-lucent layer in Helicotylenchus and Pararotylenchus species and a thick electron-lucent layer representing half of the total thickness of the cuticle in Aphasmatylenchus species; in Hoplolaimus and Aorolaimus species, two layers are present: a thin electron-dense layer consisting of densely packed osmophilic corpuscules and a thick electron-lucent layer. Intracuticular canals previously described in other genera in the subfamily occur in all species studied and may be considered constant in Hoplolaiminae; observations on lateral fields in cross section reveal a variability of their shape and of the deepness of incisures. Three major groups in the subfamily may be distinguished based on ultrastructure and relative thickness of the layers of the cuticle: i) Hoplolaimus, Scutellonema and Aorolaimus, ii) Pararotylenchus and Helicotylenchus, iii) Aphasmatylenchus and Rotylenchus. Cuticle ultrastructure in Hoplolaiminae appears totally different from that observed in other taxonomic groups of the order Tylenchida.

Isolation by ConA binding of haustoria from different rust fungi and comparison of their surface qualities

Rust haustoria isolated from infected leaf tissue strongly bind to ConA. This property was exploited to purify them by affinity chromatography on a ConA-Sepharose macrobead column. Haustoria were obtained with more than 90% purity and yields of up to 50%. Binding of haustoria to the column was partially inhibited by a ConA-specific sugar, methyl α-D-mannopyranoside. Compared to ConA,Lens culinaris agglutinin and wheat germ agglutinin were less efficient affinity ligands. Using ConA-Sepharose, rust haustoria from a variety of sources could be isolated with equal efficiency, indicating that they have similar carbohydrate surface properties. The haustoria maintained their typical shape after the isolation procedure, which suggests a rather rigid wall structure. The morphology of haustoria was characteristic both for a given species and the nuclear condition of the rust mycelium. Electron microscopy of isolated haustoria revealed an intact haustorial wall surrounded by a fibrillar layer presumably derived from the extrahaustorial matrix. The matrix thus appears to represent a layer with gel-like properties which is rich in ConA-binding carbohydrates and connected to the haustorial wall but not to the host-derived extrahaustorial membrane.

Helicoidal self-ordering of cellulose microfibrils in aqueous suspension

In many skeletal support systems of plants and animals, cellulose, chitin, and collagen occur in the form of microfibrils ordered in a chiral nematic fashion (helicoids). However, these structures remain poorly understood due to the many constituents present in biological tissues. Here we report an in vitro system that attracts by its simplicity. Only one chemical component, cellulose, is present in the form of fibrillar fragments dispersed in water. Above a critical concentration the colloidal dispersion separates spontaneously into a chiral nematic liquid crystalline phase. On drying this phase solidifies into regularly twisted fibrillar layers that mimic the structural organization of helicoids in nature.

Precapsular layer of the anterior lens capsule in early pseudoexfoliation syndrome.

We examined a possible correlation between clinical signs of early pseudoexfoliation (PSX) syndrome related to pigment dispersion and iris stroma atrophy and morphological alterations of the lens capsule. 63 anterior lens capsules (30 PSX suspects, 3 pre-PSX, 10 PSX, 20 controls) were studied by transmission and immuno-electron microscopy (TEM). In 20 PSX suspect and 3 pre-PSX capsulotomy specimens, TEM revealed a precapsular layer (0.1-11 microns in thickness) composed of microfibrils, amorphous material, and granular inclusions. The incidence of this fibrillar layer was significantly higher (p = 0.001) in PSX suspect and pre-PSX eyes than in controls (5 positive). Ultrastructural and immunohistochemical similarities of the fibrillar surface network in PSX suspect and typical PSX specimens indicate that the precapsular layer may represent a precursor of PSX. The beginning PSX process in the eye is obviously indicated by certain clinical signs.

Electron-immunocytochemical localization of laminin and type-IV collagen in the enamel organ of the rat incisor.

The presence of laminin and type-IV collagen in the basement membrane of the rat incisor enamel organ at different stages of odontogenesis was demonstrated by means of pre-embedding immunoperoxidase method.Immunoreaction for laminin and type-IV collagen was observed in the basement membrane and in the fibrillar layer attached to the membrane demarcating the inner enamel epithelium from the dental papilla. Occasional reaction products for laminin and type-IV collagen localized in the rough endoplasmic reticulum cisternae in cells of the inner enamel epithelium and dental papilla indicated cooperative production of basement membrane components. The basement membrane of differentiating ameloblasts showed reactions for laminin and type-IV collagen, though the intensity was consistently lower than that of reactions in the inner enamel epithelium. These reactions gradually became indistinct and discontinuous and disappeared entirely prior to dentin formation. At this time, positive reaction products were observed in the deep, irregular invaginations and coated pits in the distal ends of differentiating ameloblasts, indicating resorption of basement membrane components. The basement membrane-like structure appearing between the maturing ameloblasts and the enamel surface reacted positively for laminin. Occasional occurrence of reaction products in rough endoplasmic reticulum of maturing ameloblasts indicated laminin production. No reaction products for type-IV collagen, however, occurred in the basement membrane-like structure. Positive immunoreactions were observed in the basement membrane demarcating the outer enamel epithelium and the papillary layer from the dental sac. Basement membranes of blood capillaries that had penetrated into the papillary layer also showed intense immunoreaction. Reaction products were frequently seen in the perinuclear spaces and the rough endoplasmic reticulum cisternae of outer enamel epithelium cells but not of papillary layer cells.

Ultrastructure of Amastigomonas bermudensis ATCC 50234 sp. nov. : A new heterotrophic marine flagellate

The ultrastructure of a new marine heterotrophic flagellate is described. The cell is dorso-ventrally flattened and displays a steady gliding forward movement. A longitudinal groove whose lips are appressed to the substrate runs along the ventral aspect of the cell. The two heterodynamic flagella originate from the proximal end of a cytoplasmic sheath that enfolds most of the length of the anterior flagellum. The lips of this sheath are continuous with the margins of the ventral groove. The posterior trailing flagellum is held closely appressed to the cell body on one side of the ventral groove. Three bands of microtubules extend posteriorly into the cell body and are associated with the kinetosomes. The part of the surface membrane extending over the cell's dorsal and lateral aspect is five-layered, giving the semblance of two fused cell membranes. There is a fibrillar layer underneath these membranes. There is also a fibrous network whose arrays are oriented in different directions within the cytoplasm. Mitochondria have tubular cristae. Based on a comparison with previously described species of Amastigomonas, we establish the species, A. bermudensis n.sp. We further conclude that Thecamonas is a junior synonym of Amastigomonas and move the three nominal species to the latter genus.

The Transfer of Pollenkitt in Smyrnium perfoliatum (Apiaceae)

In Smyrnium perfoliatum the formation of pollenkitt within a secretory tapetum, and the subsequent breakdown of the cell organelles, is followed by the transformation of pollenkitt lumps into pollenkitt droplets. These droplets move within a locular fluid towards the pollen exine, where they enter the exine cavities after passing a fibrillar layer (remnants of the primexine-matrix) in between the tectum elements. This is followed by the fusion of pollenkitt droplets, forming a distinct layer at the bottom of the exine cavities

Surface polymers of the nematode-trapping fungus Arthrobotrys oligospora

The nematophagous fungus Arthrobotrys oligospora captures nematodes using adhesive polymers present on special hyphae (traps) which form a three-dimensional network. To understand further the adhesion mechanisms, A. oligospora surface polymers were visualized by transmission electron microscopy and characterized by chemical methods. Both traps and hyphae were surrounded by a fibrillar layer of extracellular polymers which stained with ruthenium red. The polymer layer was resistant to most of the chemicals and enzymes tested. However, part of the layer was removed by sonication in a Tris-buffer or by extraction in a chaotropic salt solution (LiCl), and the structure of the polymers was modified by treatment with Pronase E. Chemical analysis showed that the crude extracts of surface polymers removed by sonication or LiCl solution contained neutral sugars, uronic acids and proteins. Gel chromatography of the extracts revealed that the major carbohydrate-containing polymer(s) had a molecular mass of at least 100 kDa, containing neutral sugars (75% by weight, including glucose, mannose and galactose), uronic acids (6%) and proteins (19%). There was more polymer in mycelium containing trap-bearing cells than in vegetative hyphae. SDS-PAGE of the extracted polymers showed that the trap-forming cells contained at least one protein, with a molecular mass of approx. 32 kDa, not present on vegetative hyphae. Examining the capture of nematodes by traps of A. oligospora in which the layer of surface polymers was modified, or removed by chemical or enzymic treatments, showed that both proteins and carbohydrate surface polymers were involved in the adhesion process.

Cytochemical aspects of fungal penetration, haustorium formation and interfacial material in rose leaves infected by Sphaerotheca pannosa var. rosae

Fungal development and host reactions induced in rose leaf epidermal cells by the powdery mildew fungus, Sphaerotheca pannosa var. rosae , were examined on the basis of ultrastructure and of cytochemistry of chitin, pectin, and cellulose subunits. Fungal growth in the epidermis was associated with the formation of haustoria which appeared multilobed and delimited by an extrahaustorial membrane probably originating from the host plasmalemma. The extrahaustorial matrix was free of chitin as judged by the absence of labelling following incubation with a wheat germ agglutinin/ovomucoid-gold complex. Similarly, neither pectin nor cellulose was detected in this matrix, whereas these compounds were abundant in host cell walls. Various host reactions were observed at sites of fungal penetration. The formation of papillae was often associated with a restricted development of the infection peg. The most striking reaction was the occurrence of successive layers of a fibrillar material, forming a collar around the haustorial neck. Our cytochemical observations demonstrated that the collar was made of an amorphous material surrounded by fibrillar layers, the outermost ones being cellulose-rich. The possible nature and origin of this structure is discussed in relation to its possible involvement in resistance to fungal attack.

Precapsular film on the aging human lens: precursor of pseudoexfoliation?

In many older patients we observed a layer of subtle opacification on the anterior lens capsule, appearing as a ground glass film biomicroscopically. This precapsular film (PCF) could be uniform but often had radial grey lines in the mid zone, holes in the paracentral region, and was occasionally rolled up in strings. Lens capsular material obtained at cataract extraction was studied in patients with and without the film. By scanning electron microscopy the PCF appeared as a friable, incomplete fibrillar layer, with rolling of the edges suggesting loose attachment. Ultrastructurally its component fibrils were from 3-6 nm in diameter, similar to the finer fibrils in pseudoexfoliation (PSX) material. Life PSX material the layer stained positively for the elastic microfibril-associated protein, fibrillin, in a lens with radial striations. These similarities suggested that the two conditions have some relationship and that the PCF may be a precursor of PSX. Finding patches of the fibrillar network in some control patients implies that the PCF is common in patients of cataract age, though seldom detected clinically.

Artificial exudate

Despite its abundance of cell culture growth factors, serum does not enhance growth of the in vivo test system, the chick chorioallantoic membrane (CAM). Previously we have also shown that fibrinogen (Kabi) and its degradation products do not display mitogenic activity on the CAM, but have now been surprised to find stimulation of DNA synthesis (up to 2.5-fold) 18 h after application of human plasma [incorporation of [3H]thymidine control mean +/- SEM 8442 +/- 1338 dpm/CAM (n = 8); test--20 199 = 3491 (n = 6); t-test P less than 0.01)]. Plasma (platelet-rich and platelet-poor) was freshly prepared by minicolumn removal of citrate at 16 degrees C; 0.3 ml aliquots of 10% human plasma were applied to the CAM and groups were fixed at various time intervals. Cross-sections were examined histologically, including immunoperoxidase studies with antihuman fibrinogen which does not cross react with chick fibrinogen, and autoradiography with [3H]thymidine. Initially, a layer of plasma was observed on the surface of the CAM. After 1 h a condensed fibrillar layer of fibrin was formed and was still present at 6 h. This was associated with increased [3H]thymidine labelling of the surface layer of the CAM. By 18 h the fibrin had disappeared, indicating that it had been lysed by the CAM, and widespread [3H]thymidine labelling was observed. No inflammatory cell infiltrate was apparent, but marked oedema developed. Oedema alone was observed in controls receiving serum or Dulbecco buffer. Both platelet-rich and platelet-free plasma gave stimulation of mitogenic activity, implying that platelet-derived growth factor is not involved in the stimulation of the CAM.(ABSTRACT TRUNCATED AT 250 WORDS)

Initial attachment ofCandida albicans cells to buccal epithelial cells

The rapid-freezing technique was applied in association with scanning and transmission electron microscopy to observe the initial attachment (or contact) of Candida albicans cells to exfoliated human buccal epithelial cells. Low temperature scanning electron microscopy provided detailed three-dimensional morphological features of the yeast-epithelial cell association; adhesion of C. albicans cells to host cells was primarily owing to an interaction between fibrillar layer of the yeast cell wall and the membrane interdigitations of the epithelial cells. Such a particular interconnection between the two cells was confirmed by the freeze-substitution fixation for transmission electron microscopy. These results clearly demonstrate the outermost fibrillar cell wall layer of C. albicans responsible for adhesion to host cells.

Wall appositions induced by ionophore A 23187, CaCl2, LaCl3, and nifedipine in characean cells

The formation of wall appositions (plugs) by ionophore A 23187, CaCl2, LaCl3, and nifedipine was studied in mature internodal cells of characeaen algae. CaCl2 at concentrations above 10−2M induces thick fibrillar plugs without callose inNitella flexilis. InChara corallina andNitella flexilis ionophore A 23187 (1.25×10−5 to 5×10−5M) and LaCl3 (7.5×10−5 to 2.5×10−4M) cause flat appositions which contain callose and have a more granular structure. Plug formation by ionophore A 23187, CaCl2, and LaCl3 is pH-dependent and occurs beneath the alkaline regions of the cell. Nifedipine (10−4 to 10−5M) induces plugs inNitella flexilis after previous injury. These callose-containing wall appositions consist of a heterogeneous granular core which is covered by a fibrillar layer. The results of this work are compared with previous studies on wound wall formation and chlortetracycline (CTC)-induced plug formation which reveal that abundant coated vesicles occur only when a thick fibrillar wall layer is formed. Neither LaCl3 nor nifedipine inhibit the formation of CaCl2- or CTC-plugs. The unusual effects of these substances, which normally act as Ca2+ antagonists and therefore should prevent and not induce plug formation, are discussed. It is suggested that La3+ mimicks the effects of calcium and that nifedipine binding to the Ca2+ channels is altered in the alkaline regions of characean internodes and allows an influx of Ca2+.

The arrangement of collagen fibrils in the lymphatic sinus wall of the rabbit appendix.

The lymphatic sinus of the rabbit appendix was examined by scanning and transmission electron microscopy. Luminal surfaces of the sinus were lined by squamous endothelial cells, some of which, however, were blebby in surface relief due to the presence of electron dense granules in the cytoplasm. Many clusters of lymphocytes were present on the endothelial surface, mainly along the blood vessels of the trabeculae and of the sinus wall. Regional differences in collagen fibrillar arrangement of the sinus wall were demonstrated after the removal of the endothelial cells by the NaOH maceration method (OHTANI, 1987). In the upper region of the sinus and the lymphatic plexus of the internodular thymus dependent area (TDA), the fibrillar layer was a delicate network of interwoven thin fibrils. Furthermore, transmission electron microscopy revealed frequent transmural passages of lymphocytes, which suggest the existence of pores or fenestrations in the fibrillar layer of these regions. The fibrillar layer in the lateral and basal sinus was formed by densely interwoven thick bundles or sheets which consisted of thin collagen fibrils. No obvious space was seen between fibrils. These observations suggest that the upper region of the sinus and the lymphatics in the TDA may be active sites of cell migration from the reticular tissue.

Oral adhesion of yeasts

Oral adhesion of yeasts probably occurs by interaction between yeast cell adhesins and oral epithelial cell receptors. In Candida albicans mannoprotein, glucan, chitin, cell wall proteins, and lipids are possible adhesins. Mannoprotein appears as a fibrillar or floccular outermost layer in stationary-phase cells grown in sugar-rich medium. Preincubation of buccal epithelial cells (BECs) with concanavalin A inhibits adhesion, as does suppression of mannoprotein production by tunicamycin. Germ tubes adhere more easily to BECs and plastic than do blastospores. Methyl-alpha-D-mannoside may be analogous to the yeast adhesin or epithelial cell receptor because it inhibits adhesion of C. albicans to BECs. L-Fucose, N-acetyl-D-glucosamine, or D-mannose, having the same effect, may also function as epithelial cell receptors. Other factors affecting yeast adhesion may be fibronectin, hydrophobicity, s-IgA, and indigenous bacteria. Growth of yeasts to stationary phase in sugar-rich media promotes adhesion to acrylic, as do divalent cations and serum. Saliva, chlorhexidine, and Streptococcus salivarius inhibit adhesion of yeasts.

Laminin receptors on Candida albicans germ tubes

Recent evidence for the role of laminin in cell adhesion and in the pathogenesis of several bacterial infections has led us to investigate the existence of receptors for this extracellular matrix component in Candida albicans. At first, immunofluorescence demonstrated the presence of laminin-binding sites at the surface of germ tubes. Electron microscopy confirmed this result and permitted precise localization of the binding sites on the outermost fibrillar layer of the germ tube cell wall. By using 125I-radiolabeled laminin, the binding was shown to be saturable and specific, hence demonstrating characteristics of true receptors. Analysis of the data by the Scatchard equation indicated that there were about 8,000 binding sites per cell, with a dissociation constant (Kd) of 1.3 x 10(-9) M. Binding was inhibited by prior heating or trypsinization of cells. Furthermore, of the different proteins and carbohydrates tested in competition experiments, only fibrinogen greatly reduced the laminin binding. Finally, dithiothreitol and iodoacetamide treatment of germ tubes allowed us to identify the laminin receptors through analysis of this extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. Two components, of 68 kilodaltons and a doublet of 60 and 62 kilodaltons, were detected. Thus, C. albicans possesses germ tube-specific surface receptors for laminin which could mediate its attachment to basement membranes and so contribute to the establishment of candidiasis.

The Relationship between Coaggregation Properties and Surface Structures ofBacteroides intermedius

Seven out of 20 Bacteroides intermedius isolates coaggregated with two Actinomyces viscosus and four A. naeslundii strains. None coaggregated with seven other strains representing five Gram-positive oral species. Coaggregating ability was unrelated to B. intermedius serogroup, surface hydrophobicity, or morphology of fibrils or ruthenium red (RR) staining layers. The coaggregation adhesin on B. interrnedius isolates recognising A. viscosus and A. naeslundii was unaffected by serum, saliva, sugars and Congo red. It was inactivated by NaIO 4 , heat, proteinase K and, in 4/7 isolates, by trypsin but not by heat-inactivated enzymes. Coaggregation adhesins on serogroup I isolates were resistant to trypsin, and were less susceptible than those on other strains to heat and proteinase K. Cell surface hydrophobicity was unaltered by NaIO 4 and trypsin. RR staining matrices and capsules (detected by staining with Indian ink) were unaffected by trypsin, but NaIO 4 removed both. Capsules and fibrils were removed at different times by NaIO 4 and were therefore different structures. Coaggregating ability disappeared with removal of fibrils indicating the adhesin was associated with the fibrillar layer. All B. intermedius isolates carried two types of fibril. One was removed by trypsin treatment but was not involved in coaggregation. The other was not removed by trypsin or proteinase K. The proteinase K and heat-inactivatable coaggregation adhesin may be a non-structural component of these fibrils. Keywords: B. intermedius ; Coaggregation; Adhesins; Actiriomyces spp. ; Ultrastructure; Fibrils; Ruthenium red; Capsules; Hydrophobicity.