Induction of platelet aggregation after a direct physical interaction with diesel exhaust particles.

BACKGROUND: There is a proven link between exposure to traffic-derived particulate air pollution and the incidence of platelet-driven cardiovascular diseases. It is suggested that inhalation of small, nanosized particles increases cardiovascular risk via toxicological and inflammatory processes and translocation of nanoparticles into the bloodstream has been shown in experimental models. We therefore investigated the ability of diesel exhaust particles (DEP) to interact physically and functionally with platelets. METHODS: The interaction of DEP and carbon black (CB) with platelets was examined by transmission electron microscopy (TEM), whereas the functional consequences of exposure were assessed by measuring in vitro and in vivo platelet aggregation via established methods. RESULTS: Both DEP and CB were internalized and seen in proximity with the open canalicular system in platelets. DEP induced platelet aggregation in vitro whereas CB had no effect. DEP induced Ca(2+) release, dense granule secretion and surface P-selectin expression, but not toxicologic membrane disruption. Low concentrations of DEP potentiated agonist-induced platelet aggregation in vitro and in vivo. CONCLUSIONS: DEP associate physically with platelets in parallel with a Ca(2+) -mediated aggregation response displaying the conventional features of agonist-induced aggregation. The ability of DEP to enhance the aggregation response to platelet stimuli would be expected to increase the incidence of platelet-driven cardiovascular events should they be inhaled and translocate into the blood. This study provides a potential mechanism for the increased thrombotic risk associated with exposure to ambient particulate air pollution.

Structure and nucleotide-dependent changes of thick filaments in relaxed and rigor plaice fin muscle.

The myosin crossbridge array, positions of non-crossbridge densities on the backbone, and the A-band "end filaments" have been compared in chemically skinned, unfixed, uncryoprotected relaxed, and rigor plaice fin muscles using the freeze-fracture, deep-etch, rotary-shadowing technique. The images provide a direct demonstration of the helical packing of the myosin heads in situ in relaxed muscle and show rearrangements of the myosin heads, and possibly of other myosin filament proteins, when the heads lose ATP on going into rigor. In the H-zone these changes are consistent with crossbridge changes previously shown by others using freeze-substitution. In addition, new evidence is presented of protein rearrangements in the M-region (bare zone), associated with the transition from the relaxed to the rigor state, including a 27-nm increase in the apparent width of the M-region. This is interpreted as being mostly due to loss or rearrangement of a nonmyosin (M9) protein component at the M-region edge. The structure and titin periodicity of the end-filaments are described, as are suggestions of titin structure on the myosin filament backbone.

Three-dimensional structure of a vertebrate muscle Z-band: implications for titin and alpha-actinin binding.

The Z-band in vertebrate striated muscles, mainly comprising actin filaments, alpha-actinin, and titin, serves to organise the antiparallel actin filament arrays in adjacent sarcomeres and to transmit tension between sarcomeres during activation. Different Z-band thicknesses, formed from different numbers of zigzag crosslinking layers and found in different fibre types, are thought to be associated with the number of repetitive N-terminal sequence domains of titin. In order to understand myofibril formation it is necessary to correlate the ultrastructures and sequences of the actin filaments, titin, and alpha-actinin in characteristic Z-bands. Here electron micrographs of the intermediate width, basketweave Z-band of plaice fin muscle have been subject to a novel 3D reconstruction process. The reconstruction shows that antiparallel actin filaments overlap in the Z-band by about 22-25 nm. There are three levels of Z-links (probably alpha-actinin) in which at each level two nearly diametrically opposed links join an actin filament to two of its antiparallel neighbours. One set of links is centrally located in the Z-band and there are flanking levels orthogonal to this. A 3D model of the observed structure shows how Z-bands of different widths may be formed and it provides insights into the structural arrangements of titin and alpha-actinin in the Z-band. The model shows that the two observed symmetries in different Z-bands, c2 and p12(1), may be attributed respectively to whether the number of Z-link levels is odd or even.

Structure of abnormal molecular assemblies (collagen VI) associated with human full thickness macular holes.

Transversely banded deposits with an approximately 100-nm periodicity have been seen in association with a number of eye pathologies (e.g., age-related macular degeneration). Recently such aggregates have also been discovered in the cortical vitreous of a patient suffering from full thickness macular holes. The aggregates in the vitreous were of sufficient size and regularity for us to attempt 3D ultrastructural studies in the electron microscope. The molecules forming this aggregate pack in a centered tetragonal unit cell of dimensions approximately 26 x 26 x 180 nm. A real-space (r-weighted back projection) 3D reconstruction was computed. The aggregate is discussed in terms of its possible protein constituents. Collagen VI has been singled out as the most likely protein to form the aggregate. Two alternative models for the molecular packing are proposed, comprising aggregates of molecular tetramers or octamers. Understanding the structure of these abnormal banded deposits in the eye should help to throw light on the pathophysiological mechanisms of the diseases, including age-related macular degeneration, in which they occur.

A-band architecture in vertebrate skeletal muscle: polarity of the myosin head array.

Despite extensive knowledge of many muscle A-band proteins (myosin molecules, titin, C-protein (MyBP-C)), details of the organization of these molecules to form myosin filaments remain unclear. Recently the myosin head (crossbridge) configuration in a relaxed vertebrate muscle was determined from low-angle X-ray diffraction (Hudson et al. (1997), J Mol Biol 273: 440-455). This showed that, even without C-protein, the myosin head array displays a characteristic polar pattern with every third 143 A-spaced crossbridge level particularly prominent. However, X-ray diffraction cannot determine the polarity of the crossbridge array relative to the neighbouring actin filaments; information crucial to a proper understanding of the contractile event. Here, electron micrographs of negatively-stained goldfish A-segments and of fast-frozen, freeze-fractured plaice A-bands have been used to determine the resting myosin head polarity relative to the M-band. In agreement with the X-ray data, the prominent 429 A-spaced striations are seen outside the C-zone, where no non-myosin proteins apart from titin are thought to be located. The head orientation is with the concave side of the curved myosin heads (containing the entrance to the ATP-binding site) facing towards the M-band and the convex surface (containing the actin-binding region at one end) facing away from the M-band.

Partially systematic molecular packing in the hexagonal columnar phase of dogfish egg case collagen.

The collagen that forms the egg case of the dogfish Scyliorhinus canicula is stored in bulk in the female nidamental glands. Here the collagen molecules are thought to undergo a series of distinct pH-dependent liquid crystalline aggregation phase changes before assembling into the final arrangement encountered in the mature egg case. One liquid crystalline phase is hexagonal with the centres of two adjacent hexagons about 36 nm apart. We have collected tilt series of the hexagonal phase from plastic sections of the nidamental gland and have produced a three-dimensional reconstruction of the collagen arrangement of this phase. The reconstruction features axial columns of protein density lying regularly on the vertices of hexagonal cells of edge length 21 nm. Each column is connected to three nearest neighbours by irregular sheets of protein, but there appear to be preferred molecular directions at about 40 degrees to 50 degrees to the columns. The reconstruction has been interpreted in terms of known interactions of this collagen in other assemblies.

Evolution of myosin filament arrangements in vertebrate skeletal muscle.

A survey of skeletal muscles throughout craniates shows basic kinds of myosin filament arrangement, simple-lattice and superlattice, within the A-band of each sarcomere. Distribution of simple- and superlattice arrangements across a phylogeny of craniates suggests that the superlattice arrangement is primitive and that Amia and teleosts are derived in showing simple-lattice arrangements. Two taxa examined (Scyliorhinus and Acipenser) show both lattice types within the same organism implying that there is not a simple evolutionary transformation of one to the other fiber arrangement. We discuss the possible functional significance of the different lattice types. We believe that the crossbridges may have greater competition for actin binding sites in simple-lattice muscles compared to the superlattice types.

Examination of the structure of the transthyretin amyloid fibril by image reconstruction from electron micrographs.

Familial amyloidotic polyneuropathies are autosomal-dominant, inherited disorders that are characterised by the aggregation of variant proteins in a fibrillar form and by the extracellular deposition of amyloid fibrils. In familial amyloidotic polyneuropathy type I the protein constituent is a variant transthyretin molecule that has a Val to Met substitution at residue 30. Patients with this form of the disease present with sensory and motor disturbances, widespread autonomic dysfunction and in some cases, vitreous opacities. We have used amyloid material from the vitreous humours of patients homozygous for this mutation and analysed the structure of the fibrils by thin section electron microscopy and image reconstruction. Cross-sectional images of 200 different fibrils were collected and aligned, manually at first and then with an automated process that uses iterative cross-correlation. The averaged cross-section calculated produced a detailed view of the fibril substructure. The diameter of the fibrils is about 130 A. In cross-section they exhibit 4-fold symmetry with four proto-filaments, each measuring 40 to 50 A across, arranged around a central hollow core.

M-band structure, M-bridge interactions and contraction speed in vertebrate cardiac muscles.

Cardiac muscle M-band structures in several mammals (guinea pig, rabbit, rat and cow) and also from three teleosts (plaice, carp and roach), have been studied using electron microscopy and image processing. Axial structure seen in negatively stained isolated myofibrils or negatively stained cryo-sections shows the presence of five strong M-bridge lines (M6, M4, M1, M4' and M6') except in the case of the teleost M-bands in which the central M-line (M1) is absent, giving a four-line M-band. The M4 (M4') lines are consistently strong in all muscles, supporting the suggestion that bridges at this position are important for the structural integrity of the A-band myosin filament lattice. Across the vertebrate kingdom, cardiac M-band ultrastructure appears to correlate roughly with heartbeat frequency, just as in skeletal muscles it correlates with contraction speed, reinforcing the suggestion that some M-band components may have a significant physiological role. Apart from rat heart, which is relatively fast and has a conventional five-line M-band with M1 and M4 approximately equal, the rabbit, guinea pig and beef heart M-bands from a new 1 + 4 class; M1 is relatively very much stronger than M4. Transverse sections of the teleost (roach) cardiac A-band show a simple lattice arrangement of myosin filaments, just as teleost skeletal muscles. Almost all other vertebrate striated muscles, including mammalian heart muscles, have a statistical superlattice structure. The high degree of filament lattice order in teleost cardiac muscles indicates their potential usefulness for ultrastructural studies. It is shown that, in four-line M-bands in which the central (M1) M-bridges are missing, interactions at M4 (M4') are sufficient to define the different myosin filament orientations in simple lattice and superlattice A-bands. However the presence of M1 bridges may improve the axial order of the A-band.

Three-dimensional reconstruction of a simple Z-band in fish muscle.

The three-dimensional structure of the Z-band in fish white muscle has been investigated by electron microscopy. This Z-band is described as simple, since in longitudinal sections it has the appearance of a single zigzag pattern connecting the ends of actin filaments of opposite polarity from adjacent sarcomeres. The reconstruction shows two pairs of links, the Z-links, between one actin filament and the facing four actin filaments in the adjacent sarcomere. The members of each pair have nearly diametrically opposed origins. In relation to one actin filament, one pair of links appears to bind along the final 10 nm of the actin filament (proximal site) and the other pair binds along a region extending from 5 to 20 nm from the filament end (distal site). Between one pair and the other, there is a rotation of approximately 80 degrees round the filament axis. A Z-link with a proximal site at the end of one actin filament attaches at a distal site on the oppositely oriented actin filaments of the facing sarcomere and vice versa. The length of each Z-link is consistent with the length of an alpha-actinin molecule. An additional set of links located 10-15 nm from the center of the Z-band occurs between actin filaments of the same polarity. These polar links connect the actin filaments along the same direction on each side of the Z-band. The three-dimensional structure appears to have twofold screw symmetry about the central plane of the Z-band. Only approximate twofold rotational symmetry is observed in directions parallel to the actin filaments. Previous models of the Z-band in which four identical and rotationally symmetrical links emanate from the end of one actin filament and span across to the ends of four actin filaments in the adjacent sarcomere are therefore incorrect.

Computation of a three dimensional image of a periodic specimen from a single view of an oblique section.

We describe here a method for computing a three dimensional map of a periodic specimen from a single electron micrograph of an obliquely cut section. Neighbouring areas of such an image display successively the contents of the unit cell of the structure. The reconstruction procedure can be considered in two steps. The first step involves restacking of successive areas to produce an image akin to that produced by serial section reconstruction. The resolution normal to the section would, at this stage, be limited by the thickness of the section, since the micrograph represents a projection of the density in the section. However, because of the periodic nature of the specimen, the image contains redundant information, which can be used in an attempt to deconvolute the section thickness and thus produce improved resolution normal to the section. The computation can be carried out directly with the densities or more conveniently, particularly for three dimensional crystals, by using Fourier transforms. The approach, which is most powerful when the section is thin, is insensitive to the collapse of the section caused by electron irradiation. Striated muscle provides particularly suitable specimens for such analysis and we present, as examples, computed maps of the M-band of fish muscle and of insect flight muscle in rigor.

A method for monitoring the collapse of plastic sections as a function of electron dose.

We present a method for monitoring the collapse of plastic sections when irradiated in the electron microscope. The two surfaces of the section are separately coated with colloidal gold particles. The section is then tilted to an angle of 45 degrees in the microscope and a series of micrographs recorded, corresponding to increasing total electron dose. The collapse of the specimen normal to the plane of the section causes a relative movement in the image of the two sets of particles marking the two surfaces. By measuring the positions of a few gold particles on each side of the section in each exposure of the series, the collapse and also the in-plane shrinkage can be computed. The sections exhibit a rapid initial collapse, followed by a much slower phase of thinning. These effects should be taken into account when producing quantitative three-dimensional maps from tilt series of sectioned material.

Three-dimensional reconstruction from tilted sections of fish muscle M-band.

Fish muscle provides a particularly suitable specimen for studying the organization of thick filaments in vertebrate striated muscle because the filaments are arranged with a single orientation on a well ordered hexagonal lattice. The M-band consists of sets of cross-links, which join the myosin filaments in the middle of the A-band. Previous work concluded that the fish muscle M-band had the local symmetry of the dihedral point group 32. We present here a quantitative analysis of transverse sections of the M-band, using general methods developed for crystalline layers, to combine various tilted views. The three-dimensional map computed to a resolution of about 70 A confirms previous findings; it shows new features of the thick filament structure in the M-band region and provides new information on the use of three-dimensional reconstruction from sectioned biological material. The accompanying paper describes a novel technique of three-dimensional reconstruction of the fish M-band from a single view of a slightly oblique plastic section.

Three-dimensional reconstruction from a single oblique section of fish muscle M-band.

A new method of three-dimensional image reconstruction from electron micrographs is presented here, which enables a three-dimensional image to be produced from a single oblique section of a two- or three-dimensional crystal. The method, which is most powerful when the section is thin and the angle of obliquity small, has several potential advantages over the conventional method of reconstruction using tilted views of the section. In particular the accumulated electron dose on the specimen is much smaller and the reconstruction is not affected by changes in the thickness of the section during exposure to the electron beam. The method involves the solution of a generally almost singular set of linear projection equations, relating through the sectioning geometry the three-dimensional crystal density to the two-dimensional projected image density. This can be achieved most conveniently by linear least squares filtering. An application of the method to determine the structure of the M-band of fish muscle is described. The resulting map agrees well with that produced by the more conventional approach involving tilted views.

High-voltage electron microscopy of crossbridge interactions in striated muscle.

In order to investigate the geometry of the interactions which myosin molecules make with actin filaments we have studied thick (0.2--0.5 micrometer) transverse sections of striated muscles in the 1 Me V electron microscope at Imperial College. Sections obtained from fixed relaxed frog sartorius muscle and both fixed relaxed and fixed rigor insect flight muscles, show regular electron opaque features between the thick and thin filament profiles. These are thought to be the overlapping images of the many levels of myosin heads that occur in such sections. From the appearances of these images, together with studies of thin transverse sections, it appears that of the possible interactions which one myosin molecule can make, namely that its two component heads interact with the same thin filament or with two different thin filaments, it is the former interaction (both heads on the same filament) which is predominant. Nevertheless appearances have been seen similar to those expected if an interaction of one molecule with two thin filaments occurs. It is concluded that both single filament and two filament interactions can occur depending on the steric convenience of the available actin subunits, but that the single filament interaction occurs in the majority of cases in the muscle states we have studied. Finally it is shown that the myosin filament profiles seen in thick transverse sections may be a very misleading guide to thick filament structure because of the influence which the myosin crossbridge have on the appearance of the profiles.

Malignant neoplasms of the paranasal sinuses involving the skin.

Malignant neoplasms of the mucosa and minor salivary glands of the paranasal sinuses may involve the skin by direct extension. When a tumor appears on the overlying skin, these sinuses should be considered as a possible site of origin. Adenoid cystic carcinoma of the paranasal sinuses arise from minor salivary glands. They can infiltrate overlying skin and easily be confused with a primary cutaneous adenoid cystic carcinoma. Malignant melanomas of the paranasal sinuses are clinically very aggressive. They are often amelanotic, and this may lead to an incorrect histopathologic diagnosis. Hence, physical and radiological examination of the nose, mouth, and paranasal sinuses should be performed whenever a tumor appears in the overlying skin that does not have a clear cutaneous origin or whenever the primary site of a metastatic malignant melanoma is unknown.