Direct visualisation of the beta-sheet structure of synthetic Alzheimer's amyloid

Amyloid fibrils are a major pathological feature of Alzheimer's disease as well as other amyloidoses including the prion diseases. They are an unusual phenomenon, being made up of different, normally soluble proteins which undergo a profound conformational change and assemble to form very stable, insoluble fibrils which accumulate in the extracellular spaces. In Alzheimer's disease the amyloid fibrils are composed of the A beta protein. Knowledge of the structure of amyloid is essential for understanding the abnormal assembly and deposition of these fibrils and could lead to the rational design of therapeutic agents for their prevention or disaggregation. Here we reveal the core structure of an Alzheimer's amyloid fibril by direct visualisation using cryo-electron microscopy. Synthetic amyloid fibrils composed of A beta residues 11 to 25 and 1 to 42 were examined. The A beta (11-25) fibrils are clearly composed of beta-sheet structure that is observable as striations across the fibres. The beta-strands run perpendicular to the fibre axis and the projections show that the fibres are composed of beta-sheets with the strands in direct register. This observation has implications not only for the further understanding of amyloid, but also for the development of cryo-electron microscopy for direct visualisation of secondary structure.     

The fibrous system in the extracellular matrix of hydromedusae

The ultrastructure and the histochemistry of the fibrous system in the mesogloeal extracellular matrix (ECM) of two hydromedusae (Polyorchis penicillatus and Aglanlha digitale) has been examined. There is a fundamental difference in the architecture of the fibrous system between the two species. In Polyorchis, 60-150 A thick, striated fibrils with periodicities of 60-65 A form a three-dimensional network which fills in the entire ECM of outer and inner mesogloea. In the outer mesogloea vertical fibres (up to 1.8 mum in diameter) penetrate the threedimensional network and branch near the exumbrellar and subumbrellar side. These branches impinge on a dense matrix covering the exumbrellar and subumbrellar surface. In Aglantha the branches of thick vertical fibres anchor at the subumbrellar side in a dense plexus (0.2-0.3 mum in thickness) which consists of two types of fibrils (35-40 and 80-100 nm in diameter). Towards the exumbrellar side the vertical fibres branch and intermingle with a meshwork of non-striated fibrils with uniform diameter (35-40 nm). These fibrils form a laminated structure (about 1 mum in thickness) so that fibrils of each layer course in the same direction but fibrils of adjacent layers run perpendicularly to each other. The banded pattern with periodicities of 600-640 A observed in the electron microscope and by histochemical methods confirm the thick vertical fibres and their branches to be a collagen. There is also strong evidence that the laminated structure in Aglantha represents layers of collagen fibrils.     

Some observations on the collagen fibrils of the egg capsule of the dogfish, Scyliorhinus canicula

The egg capsule of the dogfish Scyliorhinus canicula is a collagenous material with a laminated, plywood (orthogonal) construction. The collagen fibrils which constitute the bulk of the egg capsule wall have a unique, highly ordered structure (Knight and Hunt, 1974; 1976, 1986; Gathercole et al., 1993) which is thought to represent a smectic A liquid crystalline phase (Knight et al., 1993). The egg capsule is extremely strong and chemically inert (Hunt, 1985). It is stored, secreted and formed by the nidamental gland (Rusaou?n 1976, 1990 a, b; Knight and Feng, 1992). During intracellular storage, secretion and fibrillogenesis, the dogfish egg capsule collagen appears to pass through a remarkable series of textures within a lyotropic liquid crystalline phase diagram (Knight et al., 1993). In the present communication, further observations on the ultrastructure of the collagen fibrils and their arrangement within the laminae of the fully-formed egg capsule are reported. The effect of tilting ultrathin sections of fibrils in the goniometer stage of a transmission electron microscope are described, demonstrating that the crystalline lattice within the fibril appeared twisted more or less regularly into a long pitch helix. Other observations indicated that some of the fibrils were in turn twisted round one another to form fibres which therefore had a coiled-coil structure. The fibres are arranged parallel to one another in the laminae which are stacked to give an orthogonal plywood construction. The effects of staining fibrils with cuprolinic blue and with tannic acid are reported. Reduction in the water content of the fibrils before fixation appeared to move some of the fibrils through the part of the lyotropic phase transition diagram converting them from smectic A to smectic C. Finally, evidence is presented that the fibrils shrank, but remarkably, still retained a longitudinally-ordered but modified, molecular arrangement even after boiling in water for periods of up to 10 min. These observations are discussed in relation to other collagens.     

Chromatin ultrastructure changes during spermatogenesis in armadillos

The chromatin fibres of the male gamete nucleus of Dasypus novemcinctus contain fibrils. Measurements indicate that the chromatin fibre diameter and the number of fibrils within the fibre increase, whereas the diameter of the fibrils decreases during spermiogenesis. It is suggested that the fibrils represent a DNA-protein complex. It is further suggested that the increased fibre diameter, and the increased number of fibrils correlates with the formation of larger fibres during the condensation of the spermatid nucleus, whereas the decrease in fibril diameter is correlated with the replacement of histones with new low molecular weight acid-soluble proteins.     

Histochemical and Ultrastructural Features of the Biceps Brachii of the African Chameleon (Chamaeleo senegalensis)

Abstract Characteristics of reptilian muscle fibres were investigated in the biceps brachii of the African chameleon, Chamaeleo senegalensis. Fibres were classified as slow and fast. These types of fibre were distinguished on the basis of histochemical staining for myofibrillar ATPase (mATPase). Fast fibres stained dark for mATPase while slow fibres stained light. The patterns of innervation of slow and fast fibres were investigated by staining nerve endings for acetylcholinesterase activity. Slow fibres have a pattern of multiple innervation, whereas fast fibres are associated with individual endplates. The organization of the myofibrils and the sarcoplasmic reticulum in slow muscle fibres from the chameleon biceps brachii was compared with that in fast fibres. Slow fibres lacked an M-line and the Z-lines were uneven. They had fibrils that were not clearly separated from each other and the sarcoplasmic reticulum was poorly developed. These features are in sharp contrast to those of fast fibres which had straight Z-lines, clear M-lines and well-developed sarcoplasmic reticulum.     

Scanning electron microscope observations on the muscle innervation of Oikopleura dioica fol (appendicularia,tunicata) with notes on the arrangement of connective tissue fibres

Critical point dried and fractured appendicularia of the species Oikopleura dioica have been examined in the scanning electron microscope. The dorsal nerve cord with ganglion cells and peripheral nerve fibres could easily be observed. Thick peripheral nerve fibres leave the nerve cord as bilateral pairs at constant intervals along the tail. Most of these fibres branch from the naked nerve cord, but some evidently originate in ganglion perikarya bulging out from the nerve cord itself. These paired peripheral nerves always have elaborate end-arborizations on the medial surface of the lateral muscle cells. They are accordingly interpreted as motor axons. Some thinner peripheral nerve fibres originate at irregular intervals from both the nerve cord and the ganglion cells. Due to the numerous extracellular fibrils that connect the bilateral layers of the epidermal fins and the muscle cells to each other, these thin nerve fibres can seldom be traced to their termination. A few ones can, however, be traced ventrally between the notochord and the muscle cells and seem to end in singular bulb-like expansions. Clusters of synaptic vesicles are present in transmission electron micrographs of such nerves, and they are accordingly believed to carry efferent impulses. The extracellular fibrils are arranged in a highly ordered pattern with thick bundles crossing the gap between the structures to be interconnected and with numerous radiating insertions on the surface of the tissues.     

Types of collagen fibres in the calcareous sponges Sycon and Leucandra

Collagen from the calcareous sponges Sycon and Leucandra has been studied in thin sections and in sheaths from decalcified spicules. Sections show abundant thin fibrils 8–15 mμ in diameter and, in Sycon juveniles, 20 mμ fibrils with a clear 50 mμ period. The sheaths are also mainly composed of the thin fibrils but giant fibres up to 130 mμ in diameter and many μ in length are also found.     

Three-dimensional ultrastructure of human tendons.

The three-dimensional ultrastructure of human tendons has been studied. Epitenon and peritenon consist of a dense network of longitudinal, oblique and transversal collagen fibrils crossing the tendon fibres. The internal structure of tendon fibres is also complex. The collagen fibrils are oriented not only longitudinally but also transversely and horizontally. The longitudinal fibrils do not run only parallel but also cross each other forming spirals (plaits). These fibril bundles are bound together by a three-dimensional collagen fibril network of endotenon. In the myotendinous junction the surface of the muscle cells form processes. A network of tendineal collagen fibrils fills the recesses between the muscle cell processes penetrating the basement membrane of these processes. This complex ultrastructure of human tendons most likely offers a good buffer system against longitudinal, transversal, horizontal as well as rotational forces during movement and activity.     

Ultrastructure of induced transitions in the chromatin organization of Drosophila polytene chromosomes

The structural changes taking place in the salivary chromosomes of Drosophila melanogaster after treatment with urea-sodium hydroxide solution were studied by light and electron microscopy. An essential effect of the treatment is the gradual disappearance of the chromosomal banding pattern due to uncoiling of the chromomeric fibrils. During this process a huge amount of very thin fibrillar network is detached from the salivary chromosomes, and the longitudinal interband fibrils become aggregated to form a distinct central axis. This gives apparent likeness to a lampbrush chromosome. Even though at the light microscope level certain regions of the axial core appear to have been lost, no signs of breaks in the linear coherence of the chromosome can be observed in the electron micrographs. Because uncoiling of the chromomeres does not interrupt the continuity of the linear fibres, these observations on induced transitions lend support to the idea that the chromomeric fibrils are to some extent independent and dissimilar as compared to the interchromomeric fibres.Dedicated to Professor Esko Suomalainen in honour of his 60th birthday on June 11, 1970.     

Interactions of collagen molecules in the presence of N-hydroxysuccinimide activated adipic acid (NHS-AA) as a crosslinking agent

The effect of crosslinking agent on pepsin-soluble bovine collagen solution was examined using N-hydroxysuccinimide activated adipic acid (NHS-AA) as a crosslinker. Electrophoretic patterns indicated that crosslinks formed when NHS-AA was added. A higher polarity level deduced from the changes in the fluorescence emission spectrum of pyrene in the crosslinked collagen solution indicated that the formation of well-ordered aggregates was suppressed. The random aggregation of collagens was also observed by atomic force microscopy (AFM). Furthermore, the association of collagens into fibrils was influenced by crosslinking. Self-assembly was suppressed at 37 °C; however, as temperature was increased to 39 °C, a small amount of NHS-AA leaded to an improvement in the ability of self-aggregation. Although more random structure was brought about by crosslinking, self-aggregation might still be promoted as temperature was increased, accompanying by the thermal stability improvement of fibrils.     

The envelopes of the nervous system of pseudoscorpions and scorpions

The present paper demonstrates that in pseudoscorpions and scorpions a compact collagen sheath consisting of fibrils with a 720 A period embedded in a neutral mucopolysaccharide surrounds the nervous system. The same material makes up the trabeculae running into the nervous elements of the ganglia and nerves. Both structures are separated from the neurons and their fibres by entire glial cells or by their processes. The neural lamella is surrounded by a thin and continuous wall of cells forming a storage-epithelium. Amoebocytes are able to circulate between the collagen fibrils of the neural lamella.     

Ultrastructural organization of the basic substance of human dermis]

The data on ultrastructural organization of the ground substance in the human dermis obtained electron histochemically are represented. Five types of ruthenium positive structures of polysaccharide origin are detected: retinal structure (I), amorfous substance (II), membranes of collagen fibrils (III) and elastic fibres (V), fine ruthenium positive streakness of collagen fibrils (IV). These structures, except fine streakness, form a united polysaccharide system of the dermis participating in maintenance of structural-functional integrity of the connective tissue (collagen-elastic) carcass of the dermis. Two mechanisms, interconnected and oppositely directed, perform this function: the buffer mechanism preventing the connective tissue fibers and collagen fibrils to approach each other, and the binding mechanism preventing the fibrils and fibers to dissociate. The reticular structure performs mainly this function at the level of fibers, and the amorphous substance does it at the level of fibrils.     

Cooperative amyloid fibre binding and disassembly by the Hsp70 disaggregase

Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α‐synuclein, huntingtin, or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70 family member, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Clustering of Hsc70 on the fibrils appears to be necessary for disassembly. Here we use atomic force microscopy to show that segments of in vitro assembled α‐synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo‐electron tomography and total internal reflection fluorescence microscopy reveal fibrils with regions of densely bound chaperones, preferentially at one end of the fibre. Sub‐stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of ~ 4 subunits per second. The observed unidirectional bursts of Hsc70 loading and unravelling may be explained by differences between the two ends of the polar fibre structure.     

Immobilization of organophosphate hydrolase on an amyloid fibril nanoscaffold: towards bioremediation and chemical detoxification

Organophosphate hydrolase has potential as a bioremediation and chemical detoxification enzyme, but the problems of reusability and stability need to be addressed to use this enzyme on an industrial scale. Immobilizing the enzyme to a nanoscaffold may help to solve these problems. Amyloid fibrils generated from insulin and crystallin provided a novel nanoscaffold for the immobilization of organophosphate hydrolase, using glutaraldehyde as the crosslinking reagent. Electrophoretic, centrifugation, and temperature stability experiments, together with transmission electron microscopy were undertaken to verify that crosslinking had successfully occurred. The resulting fibrils remained active towards the substrate paraoxon and when immobilized to the insulin amyloid fibrils, the enzyme exhibited a significant (～ 300%) increase in the relative temperature stability at 40, 45, and 50°C (as measured by comparing the initial enzyme activity to the activity remaining after heating), compared to free enzyme. This confirms that amyloid fibrils could provide a new type of nanoscaffold for enzyme immobilization.     

Electron microscopic and preparative methods for the analysis of isopod cuticle

The crustacean cuticle consists of a complex organic matrix and a mineral phase. The physical and chemical properties of the cuticle are corellated to the specific functions of cuticular elements, leading to a large variety in its structure and composition. Investigation of the structure-function relationship in crustacean cuticle requires sophisticated methodological tools for the analysis of different aspects of the cuticular architecture. In the present paper we report improved preparation methods that, in combination with various electron microscopic techniques, have led to new insights of cuticle structure and composition in the tergite cuticle of Porcellio scaber. We used thin sections of non-decalcified tergites and decalcified resin embedded material for transmission electron microscopy and scanning transmission electron microscopy. Etched sagittal planes of bulk tergite samples were analysed with field emission scanning electron microscopy. We have found a distinct distal region within the exocuticle that differs from the subjacent proximal exocuticle in the arrangement of fibres. Within this distal exocuticle chitin-protein fibrils assemble to fibres with diameters between 15 and 50 nm that are embedded in a mineral matrix. In the proximal exocuticle and the endocuticle fibrils do not assemble to fibres and are surrounded by mineral individually. Furthermore, we show that the pore canals are filled with mineral, and demonstrate that mild etching of polished sagittal cuticle surfaces reveals regions containing mineral of diverse solubility.     

Relationship between the axial periodicity and staining of collagen by the Masson trichrome procedure

Synopsis Although wide variations in the axial periodicity of collagen fibrils in tissue sections have been reported previously, the comparative study presented in this paper of the axial periodicity and the Masson trichrome staining of collagen fibres from different tensional situations demonstrates that marked variations in collagen axial periodicity can be directly correlated with the tensional state of the tissues before fixation and with their Masson trichrome staining reaction.Collagen fibres from tensional situations consistently exhibit a longer periodicity and retain the Acid Fuchsin component of the Masson trichrome procedure, whilst collagen fibres from non-tensional situations have a shorter periodicity, fail to retain the Acid Fuchsin component, and are coloured by the Light Green counterstain.     

Flight muscle ultrastructure of susceptible and refractory mosquitoes parasitized by larval Brugia pahangi.

On parasitization with larval Brugia pahangi the infected flight muscle fibres of "resistant" Anopheles labranchiae atroparvus undergo the following ultrastructural changes. The fibres become almost totally devoid of glycogen, their sarcoplasmic reticulum becomes elongate and closely associated with muscle fibrils. These fibrils degenerate and vesicles appear both within the degenerate fibril and within elements of the sarcoplasmic reticulum. Vesicles accumulate around the worm and degenerate to a uniform mass which eventually becomes melanized from its inner edge (next to the parasite) outwards. The infected flight muscle fibres of both "resistant" Aedes aegypti and "susceptible" Aedes togoi are almost totally devoid of glycogen granules, but show no other ultrastructural change from the uninfected state.     

Collagen processing, crosslinking, and fibril bundle assembly in matrix produced by fibroblasts in long-term cultures supplemented with ascorbic acid

Human foreskin fibroblasts were cultured for up to 6 weeks in medium supplemented with ascorbic acid. During this time, the cells produced an extensive new connective tissue matrix in which the accumulated collagen (mostly type I) amounted to about 0.25 mg/10(6) cells. The matrix was highly differentiated as shown by complete processing of procollagen to collagen alpha-chains and covalent crosslinking of the collagen. Alignment of collagen fibrils occurred as the fibrils were deposited between cells, and binding of adjacent fibrils to the cell surface appeared to hold the fibrils in register. Groups of aligned fibrils were subdivided into bundles by cell-surface folds. If beta-aminopropionitrile was added to the medium, collagen crosslinking was inhibited, but not collagen synthesis or fibril bundle organization. If ascorbic acid was omitted from the culture medium, the extensive new connective tissue matrix was not produced. Our results indicate that fibroblasts in long-term cultures supplemented with ascorbic acid produce a connective tissue matrix with many in vivo-like properties including supermolecular organization of collagen.     

Electron microscopic study on the elastic and elastic related fibres in the human fascia transversalis at different ages

An electron microscopic study of the elastic fibre and elastic related fibres of the fascia transversalis of the human inguinal triangle was performed in 20 male patients aged 13 to 81 a with right indirect inguinal hernia submitted to surgical repair. The 3 fibre types comprising the elastic system (oxytalan, elaunin, and elastic fibres) tend to be ordered in a precise manner and sequence among the fibrils, fibres, and collagen fibre bundles, respectively. The present findings show that with aging, there is a decrease in the oxytalan fibres and an increase in the amorphous substance of the elastic fibres. The authors concluded that the decrease in oxytalan fibres as a function of age may be responsible for alteration in the resistance of the transversalis fascia.     

Particular structure of the anterior third of the human true vocal cord.

The histological aspects of the true vocal cord mucosa change in the anterior third compared with the posterior two thirds. The anterior third is characterized by an epithelium where the ridges, marked in the posterior two thirds, are very slight or even absent. The underlying basement membrane, which is thin in the posterior two thirds, here appears particularly thick. At the ultrastructural level in this area, beneath a normally thickened basal lamina, a thick layer of finely granulated electron-dense material, interspersed with thin and randomly scattered collagen fibrils and proteoglycan filaments, is detectable. Beneath this thickened basement membrane, a layer of small undulated collagen fibril bundles with very numerous interspersed oxytalan fibres is found. The collagen fibrils, small in diameter (30-40 nm), seem to continue with the collagen fibrils of the basement membrane. In this layer numerous blood vessels with a very thick, delaminated basement membrane are also observed. The underlying area is characterized by the vocal cord ligament, composed by large compact collagen fibril bundles with interspersed elastic fibres. The particular features of the thick basement membrane, the thick-walled and delaminated vessels and the modular distribution of the elastic system together may well form the basic structure enabling the functional integration of the vocal ligament into the overlying mucosa and the underlying vocal muscle.