Localization of chitin on ultrathin sections of cysts of two ciliated protozoa,Blepharisma undulans andPseudomicrothorax dubius,using colloidal gold conjugated wheat germ agglutinin

Summary The cyst walls of the ciliatesBlepharisma undulans andPseudomicrothorax dubius were examined ultrastructurally and by postembedding labeling with wheat germ agglutinin (WGA)-gold conjugate. Different methods of fixation and embedding were performed. In all procedures, WGA-gold binds selectively to material of the cyst wall. Pretreatment of the sections with chitinase inhibits labeling. The cyst walls of both species contain 3 nm fibrils, which are supposed to be of chitinous nature. In the cyst wall ofB. undulans, several thin layers of WGA-binding fibrils are interspaced with thick layers of other material. InP. dubius, WGA-binding sites are mainly concentrated in the mesocyst, where the microfibrils appear to represent the major component. These results obtained from two phylogenetically distant species confirm that chitin synthesis is an ancestral feature of ciliated protozoa. The amount and distribution of the chitin fibrils may play an important role in the properties and functions of the wall of the resting cyst.     

Fine structure of P-protein filaments from Ricinus communis

Summary Exudate from the phloem of Ricinus communis L. was negatively stained, examined in the electron microscope, and the filamentous components compared with those in fixed, sectioned material. In the exudate, two main fibrillar components were observed. One component has a diameter of 20±0.35 (standard error) nm, the other of 14.1±0.34 nm. This second compoent has projections along its length measuring 5 by 14 nm and spaced at intervals of 6.5–10 nm. Fibrils have been found possessing characteristics of both fibril types, suggesting some structural relationship between the two, possibly an interconvertibility. Several other types of fibrils occurred less frequently in the exudate. The exudate also contains torus-shaped structures measuring 13.5–15 nm in diameter. Sections of mature sieve elements of Ricinus and Acer rubrum L. contain fibrils structurally similar to the 14-nm fibrils from the exudate of Ricinus. Ricinus exudate was also fixed and pelleted in the ultracentrifuge. Thin sections of the pellet afforded cross-sectional views of the 20-nm fibrils, and showed that these fibrils apparently have a solid core. Possible models for the structure of the 20-nm filaments are described.     

Electron microscopic observations on interband fibrils in Drosophila salivary chromosomes

Acetic alcohol squash, preparations of salivary chromosomes of Drosophila melanogaster stained with uranylacetate during fixation and dehydration were sectioned and examined with the electron microscope. At high magnification the interband regions are seen to be composed of coiled fibrils in the range of 50 Å which often seem to be arranged in pairs. Submicroscopic bodies found along the interband fibrils seem to delimit successive subunits. It is suggested that transverse coiling of the fibrils within the boundaries of each subunit leads to the formation of chromomeres.     

Effects of glycosaminoglycans and proteoglycan on the in vitro assembly and thermal stability of collagen fibrils

The effects of three glycosaminoglycans (chondroitin 6-sulfate, dermatan sulfate, and hyaluronate) and a proteoglycan on the kinetics of fibril formation and on the thermal stability of the in vitro assembled collagen fibrils, under physiological conditions of ionic strength and pH, have been examined. The glycosaminoglycans were found to influence the kinetics of collagen precipitation but not the thermal stability of the in vitro assembled fibrils. The proteoglycan was found to influence the kinetics of collagen precipitation and to reduce the thermal stability of the in vitro assembled fibrils. Comparison of the interaction occurring between chondroitin 6-sulfate and collagen under acidic conditions (0.05M acetic acid) and that occurring under physiological conditions showed that markedly different interaction products were formed under the different conditions.     

Formation of cellulose fibrils by gram-negative bacteria and their role in bacterial flocculation

Summary Exocellular fibrils, consisting of true cellulose, were found to be produced by many bacteria. These bacteria have been selected out of a large number of strains isolated from activated sludge on the basis of their flocculent growth habit in liquid medium.The amount of cellulose, present in the bacterial flocs, varied from 1.0 to 4.0%. In addition to strains isolated from activated sludge, like Pseudomonas, Achromobacter, Alcaligenes and Aerobacter, also strains of the genera Rhizobium, Agrobacterium and Azotobacter were found to give flocculent growth due to the formation of cellulose fibrils.Bacterial flocculation in pure cultures of the strains examined was mainly caused by the production of exocellular fibrils. Apparently, the formation of cellulose fibrils seems to be a common property of Gram-negative, floc-forming bacteria, and may not be restricted to Acetobacter xylinum.     

On the fine structure of the frog's rod outer segments,observed by the freeze-etching technique

Summary The fine structure of the frog's (Rana esculenta) rod outer segments was investigated by two different methods: most of the experiments were made by means of the freeze-etching technique. The replicas were then examined by electron microscopy (40,000 X).By means of a second method, rod outer segments were negatively stained prior to electron microscopy.Inspection of the electron micrographs revealed that the frog's rod outer segments seem to be built up of three groups of elongated structures interpreted as fibrils (Fäden) arranged regularly at approximately equal distances. The diameters of the fibrils are below 100 Å; they depend on the state of light adaptation and on the chemical preparation before freeze-etching. The fibrils partly cross each other. In addition, there were found four groups of approximately equal distances between the fibrils. The order of magnitude of these spacings is from about 50 Å to a few hundred Å.Negatively stained outer segments also reveal fibrils. The results are expressed in a working hypothesis consisting of two parts. It is supposed first that the core of the rod outer segment represents a three dimensional paracrystalline lattice (Raumgitter) of three different types of fibrils (d ₁, d₂, d₄). The distances between the fibrils are interpreted as the lattice constants (a ₁, a₂, a₃, a₄). A unit cell of the lattice would consist of a web (Geflecht) of two different types of fibrils (d ₁, d₂) and four layers of parallel fibrils of the third type (d ₄).It is supposed, secondly, on the basis of a volume-evaluation, that the d₁-fibrils contain rhodopsin, those of type d ₂ another protein (not rhodopsin), and fibrils of type d ₄ lipids.The working hypothesis is supported by experimental findings of other authors (obtained by negative staining and diffraction of light and X-rays).Attempts have been made to relate some electron micrographs of ultrathin sections to those of replicas. (Rosenkranz et al., 1969; Rosenkranz, 1969a.)I wish to thank Prof. Dr. H. Stieve for the interest he took in this work through critical discussions and financial support. I also wish to thank Prof. A. Ruthmann, Ph. D., for introducing me to electron microscopy and for his linguistic aid. That Prof. Dr. K. Mühlethaler, ETH Zürich, and Prof. Dr. F. Schwanitz, KFA Jülich, put their freeze-etching apparatus and electron microscope at my disposal is gratefully acknowledged. The technical assistance of Miss M. Deichmann is also acknowledged.     

The use of tannic acid for the ultrastructural visualization of hyaluronic acid

Summary This study describes a method, which makes use of tannic acid (2%) as a component of a paraformaldehyde-glutaraldehyde based fixative, to reveal the presence and ultrastructure of glycosaminoglycans in the extracellular matrix. The ultrastructure of the extracellular matrix in the stage 24 chick embryo wing is examined after fixation by several procedures. After fixation in the absence of tannic acid, the intercellular spaces contain little extracellular matrix, except for occasional fibrils (collagen?). On the other hand, when tannic acid is included in the primary fixative, the intercellular spaces contain considerable amounts extracellular matrix which includes 3±0.5 nm filaments, ±30 nm granules, as well as putative collagen fibrils. The 3±0.5 nm diameter fibrils are not observed when the limbs had been injected in ovo with Streptomyces hyaluronidase (specific for hyaluronic acid) prior to fixation. Furthermore, the 3±0.5 nm fibrils resemble authentic hyaluronic acid that had been fixed by the same procedure in the presence of tannic acid. Limbs treated with tannic acid after osmication contained only small amounts of extracellular material, which was confined largely to cell surfaces. These results demonstrate that the use of tannic acid in the primary fixative can serve as a useful method for the ultrastructural visualization of several extracellular matrix materials, including hyaluronic acid.This study was supported by NIH grant HD 05505     

Amyloid fibril formation in vitro from halophilic metal binding protein: Its high solubility and reversibility minimized formation of amorphous protein aggregations

Halophilic proteins are characterized by high net negative charges and relatively small fraction of hydrophobic amino acids, rendering them aggregation resistant. These properties are also shared by histidine‐rich metal binding protein (HP) from moderate halophile, Chromohalobacter salexigens, used in this study. Here, we examined how halophilic proteins form amyloid fibrils in vitro. His‐tagged HP, incubated at pH 2.0 and 58°C, readily formed amyloid fibrils, as observed by thioflavin fluorescence, CD spectra, and transmission or atomic force microscopies. Under these low‐pH harsh conditions, however, His‐HP was promptly hydrolyzed to smaller peptides most likely responsible for rapid formation of amyloid fibril. Three major acid‐hydrolyzed peptides were isolated from fibrils and turned out to readily form fibrils. The synthetic peptides predicted to form fibrils in these peptide sequences by Waltz software also formed fibrils. Amyloid fibril was also readily formed from full‐length His‐HP when incubated with 10–20% 2,2,2‐trifluoroethanol at pH 7.8 and 25°C without peptide bond cleavage.     

The C-terminal extrahelical peptide of type I collagen and its role in fibrillogenesis in vitro

The formation in vitro of fibrils from type I acid-soluble calf skin collagen has been studied before and after removal of the extrahelical peptides with carboxypeptidase and with pepsin. Turbidimetric studies show that the mechanism of fibril growth in undigested collagen is similar to that in pepsin-digested collagen; following carboxypeptidase digestion, however, a different growth mechanism was apparent. The two mechanisms have been further characterized by electron microscopy. In the course of formation of fibrils from undigested collagen, “early fibrils” (short D-periodic fibrils that have both ends visible) occurred in the lag phase under the precipitating conditions employed here. After pepsin or carboxypeptidase digestion of the collagen no “early fibrils” were seen. In carboxypeptidase-digested collagen, lateral assembly was inhibited; after pepsin digestion, linear assembly was inhibited. Complete removal of the extrahelical peptides prevented fibril formation under the conditions used here. Electron-optical examination of segment-long-spacing (SLS) dimers established a more complete removal of the C-terminal peptide after carboxypeptidase digestion than after pepsin digestion. Analyses of staining patterns of SLS dimers and fibrils from undigested and digested samples showed that the C-terminal peptide in SLS crystallites and fibrils formed from undigested collagen is in a condensed conformation. A proposed conformation, in which condensation occurs predominantly in a hydrophobic region at the proximal end of the C-terminal peptide, is discussed in terms of a dual role for the C-terminal peptide in fibrillogenesis. One role, shared with the N-terminal peptide, is to participate in interactions between the 4D-staggered molecules leading to the formation of linear aggregates; the other is to participate in interactions between these linear aggregates giving rise to D-periodic aggregates and lateral (as well as linear) growth.     

Extracellular protein fibrils in Chrysochromulina breviturrita (Prymnesiophyceae) and their serological relationship to fungal fimbriae

An extensive network of extracellular fibrils was revealed by negative staining in the greenish gold algal flagellate, Chrysochromulina breviturrita. These fibrils were of uniform diameter (4–5 nm), sometimes exceeding 5 m in length. In addition there were short, narrower fibrils (2–3 nm) on the surface of the flagella. Six protein bands were isolated from spent culture medium by SDS-PAGE and one of 80,000 Da was found to polymerize after dialysis into 4–5 nm fibrils identical to those found on the cell surface. Two other proteins of 58,000 Da and 65,000 Da also formed 4–5 nm fibrils but these were either rare or of a shorter length and different appearance. An antiserum directed against the surface 7 nm fibrils (fimbriae) of fungi agglutinated cells of C. breviturrita and some other Prymnesiophyceae and Chrysophyceae, but did not agglutinate cells of algal species in other groups. Immunofluorescence and protein A gold labelling confirmed that antigens related to fungal fimbriae were present on the surface of cells of C. breviturrita. Only the 80,000 and 58,000 Da proteins labelled heavily following protein A gold labelling. Some individual 4–5 nm fibrils labelled with gold were observed in the material prepared from the 80,000 Da band. These results therefore establish that C. breviturrita produces a surface network of fibrils that are serologically related to the fimbriae of fungi, and suggest a previously unrecognized relationship between members of the Prymnesiophyceae, Chrysophyceae and fungal groups.     

Distinctive tracheid microstructure in stems of Victoria and Euryale (Nymphaeaceae)

Scanning electron microscopy (SEM) photographs of thick sections from liquid‐preserved stems of Victoria cruziana and Euryale ferox show accretions of coarse fibrils on pit membranes of tracheids. The first‐deposited fibrils are randomly orientated; on top of them (facing the tracheid lumina) are axially orientated coarse fibrils. The two systems are interconnected. Axially orientated fibrils were more extensively observed in Euryale than in Victoria and tips of fibrils in Euryale extend over the pit apertures onto secondary wall surfaces. Tracheid–parenchyma interfaces bear rudimentary coarse fibrils on the tracheid side. End walls of Victoria tracheids have highly porose pit membranes, thinner and less complex than those of the lateral intertracheid walls. The structures reported in Victoria and Euryale are consistent with those concurrently reported for stems of other Nymphaeaceae. Although also present in Cabombaceae, the coarse fibrils are otherwise not reported for stems of angiosperms and are not yet reported in roots of any species. Pit membrane remnants in perforation plates of various woody dicotyledons represent a nonhomologous phenomenon. The accretions of coarse fibrils in stem tracheids of Nymphaeaceae do not appear to enhance conduction, although they do contain porosities interconnecting tracheids. Removal of pit membrane remnants from perforation plates of primitive dicotyledon woods by hydrolysis does, on the contrary, suggest conduction enhancement. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 52–57.     

THE CELL WALL OF PYROCYSTIS SPP. (DINOCOCCALES)1,2

The cell of Pyrocystis spp. is covered by an outer layer of material resistant to strong acids and bases. Internal to this layer much of the cell wall is composed of cellulose fibrils. The presence of cellulose fibrils was established by staining raw and ultra-violet–peroxide-cleaned cell walls and by combining X-ray diffraction spectroscopy with electron microscope observation. Carbon replicas of freeze-etched preparations and thin sections of P. lunula walls show outer layers, inside them ca. 24 layers of crossed parallel cellulose fibrils (4–5 nm thick, ca. 12 nm wide), then a region of smaller (ca. 6–12 nm diameter) fibrils in a disperse texture, and then the plasma membrane. Cellulose fibrils in the parallel texture are constructed of 3–5 elementary fibrils ca. 3 nm in diameter. Walls of P. fusiformis and P. pseudonctiluca also have cellulose fibrils in a crossed parallel texture similar to those of P. lunula. The Gymnodinium-type swarmer from lunate P. lunula appears to have a cell wall ultrastructure typical of other “naked” dinoflagellates.     

Inhibitors of islet amyloid polypeptide fibrillogenesis, and the treatment of type-2 diabetes

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. Although it may be a secondary event in the etiology of diabetes, the accumulation of insoluble IAPP fibrils is considered to be a primary cause of β-cell failure in affected individuals. A possible means of inhibiting this process is through the use of small peptides that bind to IAPP and prevent fibril polymerization. This approach has been examined using a series of overlapping hexamers that target the known amyloidogenic regions of IAPP. Peptides were examined usingin vitroassays and active inhibitors were identified by their ability to prevent amyloid-related conformational transitions and IAPP aggregation. Fragments such as those corresponding to the IAPP-derived sequences, SNNFGA (residues 20–25) and GAILSS (residues 24–29), were potent inhibitors ofβ-sheet folding and amyloid fibril formation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP significantly decreased the density of fibrils and any remaining structures displayed altered morphology. In some, but not all cases, inhibition of amyloid fibrils also correlated with an ability to reduce IAPP-mediated cytotoxicity as determined in cell culture studies. The results from these studies suggest that these two peptide inhibitors differ in their mechanisms of action possibly due to unique interactions with the full-length IAPP molecule. These inhibitors form the basis of a therapeutic strategy to prevent amyloid accumulation leading to improved islet survival and a potentially novel treatment for type-2 diabetes.     

The predominant roles of the sequence periodicity in the self‐assembly of collagen‐mimetic mini‐fibrils

Collagen fibrils represent a unique case of protein folding and self‐association. We have recently successfully developed triple‐helical peptides that can further self‐assemble into collagen‐mimetic mini‐fibrils. The 35 nm axially repeating structure of the mini‐fibrils, which is designated the d‐period, is highly reminiscent of the well‐known 67 nm D‐period of native collagens when examined using TEM and atomic force spectroscopy. We postulate that it is the pseudo‐identical repeating sequence units in the primary structure of the designed peptides that give rise to the d‐period of the quaternary structure of the mini‐fibrils. In this work, we characterize the self‐assembly of two additional designed peptides: peptide Col877 and peptide Col108rr. The triple‐helix domain of Col877 consists of three pseudo‐identical amino acid sequence units arranged in tandem, whereas that of Col108rr consists of three sequence units identical in amino acid composition but different in sequence. Both peptides form stable collagen triple helices, but only triple helices Col877 self‐associate laterally under fibril forming conditions to form mini‐fibrils having the predicted d‐period. The Co108rr triple helices, however, only form nonspecific aggregates having no identifiable structural features. These results further accentuate the critical involvement of the repeating sequence units in the self‐assembly of collagen mini‐fibrils; the actual amino acid sequence of each unit has only secondary effects. Collagen is essential for tissue development and function. This novel approach to creating collagen‐mimetic fibrils can potentially impact fundamental research and have a wide range of biomedical and industrial applications.     

Cucullosporella mangrovei, ultrastructure of ascospores and their appendages

The ultrastructure ofCucullosporella mangrovei ascospores is described. Mature ascospores possess two wall layers, an outer electron-dense episporium and an innermost tripartite mesosporium. Episporial elaborations form electrondense spore wall ornamentations from which extend fibrils that may constitute a highly hydrated exosporium which was not visualised at either the scanning electron microscope or light microscope level. Ascospores possess a hamate appendage at each pole which unfolds in seawater to form a long thread. Ultrastructurally the polar appendage comprises folded fibro-granular electron-dense material and fine fibrils. The fibrils form a matrix around and within the fibro-granular appendage and around the entire unreleased ascospore. These fibrils have not been observed associated with the ascospore appendages in other species of the Halosphaeriales and are a discrete and new appendage component. The fibro-granular appendage and fibrils are bounded by the outer delimiting membrane which is absent around released ascospores. The nature of the spore appendage is compared with that of other marine and freshwater ascomycetes and the taxonomic assignment of the species is discussed.     

EGCG disaggregates amyloid-like fibrils formed by Plasmodium falciparum merozoite surface protein 2

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically unstructured and forms amyloid-like fibrils in solution. As this propensity of MSP2 to form fibrils in solution has the potential to impede its development as a vaccine candidate, finding an inhibitor that inhibits fibrillogenesis may enhance vaccine development. We have shown previously that EGCG inhibits the formation of MSP2 fibrils. Here we show that EGCG can alter the β-sheet-like structure of the fibril and disaggregate pre-formed fibrils of MSP2 into soluble oligomers. The fibril remodelling effects of EGCG and other flavonoids were characterised using Thioflavin T fluorescence assays, electron microscopy and other biophysical methods.     

Cartilage Fibrils of Mammals are Biochemically Heterogeneous: Differential Distribution of Decorin and Collagen IX

Cartilage fibrils contain collagen II as the major constituent, but the presence of additional components, minor collagens, and noncollagenous glycoproteins is thought to be crucial for modulating several fibril properties. We have examined the distribution of two fibril constituents—decorin and collagen IX—in samples of fibril fragments obtained after bovine cartilage homogenization. Decorin was preferentially associated with a population of thicker fibril fragments from adult articular cartilage, but was not present on the thinnest fibrils. The binding was specific for the gap regions of the fibrils, and depended on the decorin core protein. Collagen IX, by contrast, predominated in the population with the thinnest fibrils, and was scarce on wider fibrils. Double-labeling experiments demonstrated the coexistence of decorin and collagen IX in some fibrils of intermediate diameter, although most fibril fragments from adult cartilage were strongly positive for one component and lacked the other. Fibril fragments from fetal epiphyseal cartilage showed a different pattern, with decorin and collagen IX frequently colocalized on fragments of intermediate and large diameters. Hence, the presence of collagen IX was not exclusive for fibrils of small diameter. These results establish that articular cartilage fibrils are biochemically heterogeneous. Different populations of fibrils share collagen II, but have distinct compositions with respect to macromolecules defining their surface properties.     

Electron Microscopic Studies on the Intra-flagellar Structures of Trypanosomes*

SYNOPSIS. The intraflagellar structure (IFS) of the flagella of Trypanosoma brucei was examined on the basis of ultrathin sections in various planes. The IFS is composed of filaments approximately 50 A thick. These filaments seem to be identical with the protofilaments found earlier to be the basic elements of the contractile flagellar fibrils. The fibrillar system is firmly connected with the IFS and the latter is attached to the flagellar membrane by filaments. The lattice-like appearance of the IFS is caused by longitudinal and oblique filaments running in different planes. The structure of this network is discussed in detail. The IFS may serve as an abutment for the contractile flagellar fibrils.     

Microfibrils: a constitutive component of reticular fibers in the mouse lymph node

Summary Fibrous components other than collagen fibrils in the reticular fiber of mouse lymph node were studied by electron microscopy. Bundles of microfibrils not associated by elastin and single microfibrils dispersed among collagen fibrils were present. The diameter of the microfibrils was 13.29±2.43 nm (n=100). Elastin-associated microfibrils occurred at the periphery of the reticular fiber. Elastin was enclosed by microfibrils, thus forming the elastic fiber, which was clearly demonstrated by tannic acid-uranyl acetate staining. In the reticular fiber of lymph nodes, the elastic fiber consisted of many more microfibrils and a small amount of elastin. These microfibrils, together with the collagen fibrils, may contribut to the various functions of the reticular fibers.     

An electrostatic model for collagen fibrils. The interaction of reconstituted collagen with Ca++, Na+, and Cl−

A model for the electrostatic properties of hydrated collagen fibrils, based on the concept of a “penetrable” protein, has been evaluated through studies of collagen fibrils that had been chemically modified to change their electrostatic properties,. A value of 0.28 ± 0.07 ml/g was found for the intrafibrillar space sterically inaccessible to a molecule that had an equivalent spherical radius of 4.5 Å. The net intrinsic charge on reconstituted collagen is +14 mol/mol under physiological conditions, but decreases, at constant pH, with ionic strength. A value of 7.1 for the pK of the histidine and α-amino groups in reconstituted collagen was obtained through the application of the electrostatic model to this effect. The values obtained for calcium binding parameters for collagen fibrils, under solution conditions in which the nonspecific electrostatic properties of collagen fibrils were eliminated (3–5 M tetramethyl ammonium chloride), were in agreement with values obtained in 0.16 M NaCl solutions calculated through the use of the electrostatic theory. These are 0.73 ± 0.23 and 56.2 ± 12.3 sites per molecule with intrinsic association constants of 1101 ± 386 and 21.4 ± 5.2 M^?1, respectively. The model also predicts that an average 4-mV potential difference exists between the reconstituted collagen fibrils and physiological solutions, and that collagen fibrils under such conditions have piezoelectriclike properties. The pattern of interaction of ions with collagen fibrils is such that an allosteric mechanism for the catalytic step in the mineralization of collagen is a possibility.