Layer Of Filaments Research Articles

Surface-reactive biomaterials in osteoblast cultures: An ultrastructural study

The tissue/biomaterial interface reactions of three biomaterials selected as candidates for hard tissue replacement were studied at the electron microscopical level after incubation with enzymatically isolated rat bone cells. An electron-dense layer was routinely observed between hydroxyapatite, coral, cytodex polymer and the neighbouring cells. This layer was visible before bone formation occurred, and was collagen free. The ultrastructural features revealed a needle-shaped filamentous layer continuous with coral material, whereas hydroxyapatite or cytodex/ tissue interface was granular in appearance. These different structures may indicate reactive surfaces, depending on the composition of the substrate.

Ultrastructure of Giardia duodenalis trophozoites group from hamster: some relevant aspects

Trophozoites of Giardia duodenalis group obtained from fragments or scratched of hamster's mucosa were examined by transmission electron microscopy. The fine structure of the trophozoites are presented and compared with those described for other animals. Some of the trophozoites present the cytoplasm full of glycogen, rough endoplasmic reticulum-like structures and homogeneous inclusions not enclosed by membranes, recognized as lipid drops, which had not been observed in Giardia from other animals. The adhesive disk is composed of a layer of microtubules, from which fibrous ribbons extend into the cytoplasm; these ribbons are linked by layer of cross-bridge filaments that shows an intermediary dense band, described for the first time in this paper. The authors regard this band as the result of the cross-bridge filaments slinding in the medium region between adjacent fibrous ribbons, and suggest a contractile activity for them. The role of the adhesive disk on the trophozoite mechanism of attachment to host mucosa is also discussed.

A Unique Fibrillar Coat on the Surface of Migrating Human Primordial Germ Cells.

Primordial germ cells (PGCs) were studied by electron microscopy in human embryos at 27 and 30 days of gestation. PGCs were mainly found in the hindgut epithelium, some at the stage of separation from the endoderm, and others in migration through the mesenchyme of the dorsal mesentery between the primitive intestinal wall and the coelomic epithelium, including the coelomic angle. A few PGCs were still located in the endodermal epithelium. Before the process of separation and migration, PGCs appeared clearly different from neighboring somatic cells (endodermal, mesenchymal and epithelial cells of the hindgut and the coelomic layer). PGCs located in the endodermal epithelium were large and showed an irregular cell body often provided with filopodia-like processes that contacted somatic cells to form small areas of focal junctions. As a rule, PGCs possessed a large, round nucleus containing one or two conspicuous nucleoli. Lipid droplets, abundant glycogen particles, ribosomes and mitochondria were often observed in their cytoplasm. A detailed ultrastructural analysis revealed a delicate fibrillar coat frequently present on the free surface of PGCs during their migratory phase through the mesenchyme of the dorsal mesentery. This surface coat, likely corresponding to a glycocalyx, appeared as a particular, conspicuous filamentous layer of 30 nm thickness, mainly in the leading pseudopodial projections of PGCs migrating toward their target. The surface coat of PGCs can be associated with the binding sites of specific macromolecular components of the extracellular matrix, including fibronectin, and hence plays a role in PGCs recognition and migration during this special embryonic phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic X-ray diffraction of skeletal muscle contraction: Structural change of actin filaments

The X-ray diffraction pattern recorded during contraction shows that the force generation of a muscle proceeds upon interaction of the actin and myosin heads in the incommensurate structural framework of the thin and thick filaments. In this molecular framework the binding of myosin heads to actin filaments is thought to occur on a random basis. Such an actomyosin structure would not produce constructive interference between scattered X-rays from the bound heads and the thin filaments. The characteristic intensity changes of the thin filament layer lines that occur during contraction suggest strongly that the actin structure is varied by interaction with the myosin heads, in a manner that is quite different from that in the rigor state. Variability of the time courses of the intensity changes of the various layer lines indicates that structural change within the thin filament does not take place uniformly and that some different structural processes are involved during contraction At the plateau of isometric contraction, the thin filament structure as a whole assumes a more four-stranded nature due to the changes in the actin structure and tropomyosin position. Our present results imply that the changes of actin structure induced by interaction with myosin heads would be responsible for the regulation as well as force generation in muscle contraction.

Light- and electron-microscopic observations on the relationship between prelampbrush oocytes and surrounding granulosa cells in the laying Japanese quail (Coturnix coturnix japonica)

Transmission electronmicroscopic (TEM) observations demonstrated that the most superficial region of quail oocytes during the prelampbrush stage differs locally from the deeper ooplasm and is an active zone which forms exooplasmic cones, ridges or knob-like protrusions in the direction of/or in the granulosa cells. This exooplasm, in which no mitochondria were seen, is separated from the endooplasm, by a narrow interrupted filamentous layer. Using a lipid-preserving method of fixation, morphological evidence was found for the transport of lipid material from the granulosa cells into the exooplasm of the oocyte. Open intercellular bridges between exooplasm and granulosa cell cytoplasm were also seen. Differences between the electronmicroscopic aspect of clear and dark granulosa cells have been described.

A nonvirulent mutant of Listeria monocytogenes does not move intracellularly but still induces polymerization of actin

Listeria monocytogenes has the capacity to penetrate and multiply within professional and nonprofessional phagocytic cells, such as the Caco-2 human enterocytelike cell line. It was shown recently that shortly after listeriae have been phagocytosed, the phagosomal membrane is dissolved, probably by the action of the bacterial cytolysin listeriolysin O. The listeriae, which are then lying obviously free in the cytoplasm, become surrounded by a coat of actin filaments within a few hours. Once formed, this layer of actin filaments is reorganized in an as yet unknown way to form polar tails, which seem to be associated to the generation of listerial movement inside the cytoplasm and in intercellular spread. By using transposon Tn916 mutagenesis, a bank of L. monocytogenes mutants was generated and subsequently screened by the plaque assay system in order to select an intracellular, nonmotile mutant of L. monocytogenes. One such mutant was identified. This mutant, called L. monocytogenes M117 Imt- (for intracellular motility), like the wild type, induced actin polymerization but was not able to rearrange the actin coat to generate movement and as a result remained entrapped within the actin cloud. In a mouse virulence assay, this strain was significantly reduced in virulence. L. monocytogenes M117 is the first example to date of a Listeria mutant which is still hemolytic and invasive but reduced in virulence.

Structural basis for mechanical transduction in the frog vestibular sensory apparatus: I. The otolithic membrane

The mechanical coupling of the otoliths to the hair cell sensory stereocilia at the surface of the vestibular sensory epithelium is mediated by two layers of extracellular matrix, each one with a specific role in the mechanical transduction process. The first is a rigid layer in direct contact with the otholithic mass and is known as the otolithic membrane or gelatin membrane. This structure consists of a dense, randomly cross linked filament network that uniformly distributes the force of inertia of the non-uniform otolithic mass to all stereocilia bundles. The second layer formed by a columnar organization of filaments secures the otolithic membrane above the surface of the epithelium. The long columnar filaments are organized in parallel to the stereocilia bundles and are anchored to the apical surface of the supporting cells. The zonula adherens at the apical region of each supporting cell displays a thick polygonal bundle of actin filaments forming at the surface of the epithelium a transcellular honeycomb organization that provides mechanical ground support for the columnar filament layer. The dominant aspect of this columnar filament layer indicates that it may also have an important role in attenuating the force of inertia of the large otolithic mass during acceleration, screening stresses that would be directed to an effective bending of the stereocilia bundles.

Role of Cytokeratin Intermediate Filaments in Transhepatic Transport and Canalicular Secretion

The role of cytokeratin filaments in the function of hepatocytes was investigated using a nickel-treated hepatocyte in vitro model. Cytokeratin intermediate filaments were selectively dissociated from the cell cortex by nickel treatment. Cytokeratins and ubiquitin were observed using immunofluorescence and immunoelectron microscopy. Hepatocytic function was assessed by visualizing uptake, transhepatic transport and secretion of fluorescein diacetate and horseradish peroxidase into the bile canaliculi. In control primary cultures, most of the bile canaliculi were surrounded by an inner layer of actin filaments and an outer pericanalicular sheath of cytokeratin filaments and microtubules. The cytoplasmic distribution of ubiquitin was diffuse and particulate. After treatment with NiCl2 (150 micrograms/ml) for 24 hr, the cytokeratin filaments and desmoplakin became focally detached from the cell cortex and retracted to form an aggregate around the nucleus. These aggregates were associated with intense ubiquitin immunoreactivity. Only a few attachments of the cytokeratin filaments to the cell cortex remained. F-actin remained attached to the cell cortex in the areas where the cytokeratin filaments had become detached. The pericanalicular sheath of cytokeratin filaments and the bile canaliculi disappeared and actin was dispersed over the entire cell periphery. Fluorescein diacetate secretion and horseradish peroxidase uptake were almost completely absent in the hepatocytes treated with nickel. The effects of nickel persisted 24 hr after its removal from the medium. It is concluded that cytokeratin intermediate filaments play a critical role in the formation of the bile canaliculus, secretion of fluorescein diacetate and uptake of horseradish peroxidase. Further, our study indicates that cytokeratin ubiquitination occurs during collapse and aggregation of the cytokeratin filaments. The formation of cytokeratin-ubiquitin conjugates during aggregation suggests a role of ubiquitin in the control of cytokeratin organization in hepatocytes in the response to cell stress.

Release of sedimentary nitrogen and phosphorus in polder ditches of a low-moor peat area

The release of N and P from the sediment of two ditches, one (A) dominated by filamentous algae and the other (B) by water-lilies, was estimated by core and enclosure experiments. The release rates for ditch A tended to be higher than those for ditch B. Sediment cores covered by a filamentous algae layer released about 1.5 times more N and P than those from which the layer had been removed. During the incubation of the cores in the dark at 20°C for 2–3 weeks, about 10% of the N in the filamentous algae layer was mineralized. The mineralization could be described as a first-order reaction with a rate constant of about 0.2 d−1. On average the cores of ditches A and B released about 40 mg mineral N and 3 mg.m−2.d−1 soluble reactive phosphorus. Defining the release from the sediment in the enclosures as the net increase of N and P in the water phase and in the vegetation minus the input, a negative net release,i.e. net accumulation of N and P in the sediment, was found over the summer half of the year. The negative values were due to the significant N and P input, resulting from pumping ditch water into the enclosures in order to compensate for downward seepage. From the enclosure experiments a downward seepage rate of 14 mm.d−1 and an external load of about 6 g.m−2 total N and 0.6 g.m−2 total P during the summer half of the year —i.e. 33 mg.m−2.d−1 N and 3 mg.m−2.d−1 P. respectively — was calculated for the ditches. Tentative gross release rates — based on the sum of the positive net release of N and P into the water phase over 1–2 weeks intervals and the net increase of N and P in the vegetation — converted to 20°C and allowing for underestimation of the primary production by a factor of 5, amounted to 58 mg mineral N and 7 mg.m−2.d−1 soluble reactive phosphorus during the summer half of the year. Combining the rates estimated by cores and enclosures and converting them to rates at the mean water temperature during the summer half of the year, the release of mineral N and soluble reactive phosphorus roughly amounted to 40 and 4 mg.m−2.d−1, respectively. The release rates as well as the external load indicated a relatively low eutrophication of the ditches.

High-Resolution Electron Microscopic Evidence for the Filamentous Structure of the Cyst Wall in Giardia muris and Giardia duodenalis

High-resolution morphological studies of the cyst wall of Giardia spp. were performed using low-voltage scanning electron microscopy (LVSEM) and transmission electron microscopy (TEM). The cyst wall was composed of membranous and filamentous layers. The membranous layer consisted of an inner and an outer cyst membrane separated by a thin layer of cytoplasm. The filamentous layer contained individual filaments that ranged from 7 to 20 nm in diameter when measured by LVSEM, formed a dense meshwork with branches or interconnections, and were occasionally arranged on the surface in whorled patterns. Cysts of Giardia muris from mice, Giardia duodenalis from dogs, pigs, voles, beavers, muskrats, and humans, and Giardia psittaci from a bird (parakeet), possessed an essentially identical wall composed of filaments. Inducement of excystation in viable Giardia cysts produced a dramatic increase in the interfilament spacing over an entire cyst, but none was observed in heat-killed or chemically fixed control cysts. These results demonstrated that the cyst wall of Giardia spp. was composed of a complex arrangement of filaments, presumably formed during the process of encystment.

Three-dimensional image reconstruction of insect flight muscle. II. The rigor actin layer.

The averaged structure of rigor cross-bridges in insect flight muscle is further revealed by three-dimensional reconstruction from 25-nm sections containing a single layer of thin filaments. These exhibit two thin filament orientations that differ by 60 degrees from each other and from myac layer filaments. Data from multiple tilt views (to +/- 60 degrees) was supplemented by data from thick sections (equivalent to 90 degrees tilts). In combination with the reconstruction from the myac layer (Taylor et al., 1989), the entire unit cell is reconstructed, giving the most complete view of in situ cross-bridges yet obtained. All our reconstructions show two classes of averaged rigor cross-bridges. Lead bridges have a triangular shape with leading edge angled at approximately 45 degrees and trailing edge angled at approximately 90 degrees to the filament axis. We propose that the lead bridge contains two myosin heads of differing conformation bound along one strand of F-actin. The lead bridge is associated with a region of the thin filament that is apparently untwisted. We suggest that the untwisting may reflect the distribution of strain between myosin and actin resulting from two-headed, single filament binding in the lead bridge. Rear bridges are oriented at approximately 90 degrees to the filament axis, and are smaller and more cylindrical, suggesting that they consist of single myosin heads. The rear bridge is associated with a region of apparently normal thin filament twist. We propose that differing myosin head angles and conformations consistently observed in rigor embody different stages of the power stroke which have been trapped by a temporal sequence of rigor cross-bridge formation under the constraints of the intact filament lattice.

Three-dimensional image reconstruction of insect flight muscle. I. The rigor myac layer.

We have obtained detailed three-dimensional images of in situ cross-bridge structure in insect flight muscle by electron microscopy of multiple tilt views of single filament layers in ultrathin sections, supplemented with data from thick sections. In this report, we describe the images obtained of the myac layer, a 25-nm longitudinal section containing a single layer of alternating myosin and actin filaments. The reconstruction reveals averaged rigor cross-bridges that clearly separate into two classes constituting lead and rear chevrons within each 38.7-nm axial repeat. These two classes differ in tilt angle, size and shape, density, and slew. This new reconstruction confirms our earlier interpretation of the lead bridge as a two-headed cross-bridge and the rear bridge as a single-headed cross-bridge. The importance of complementing tilt series with additional projections outside the goniometer tilt range is demonstrated by comparison with our earlier myac layer reconstruction. Incorporation of this additional data reveals new details of rigor cross-bridge structure in situ which include clear delineation of (a) a triangular shape for the lead bridge, (b) a smaller size for the rear bridge, and (c) density continuity across the thin filament in the lead bridge. Within actin's regular 38.7-nm helical repeat, local twist variations in the thin filament that correlate with the two cross-bridge classes persist in this new reconstruction. These observations show that in situ rigor cross-bridges are not uniform, and suggest three different myosin head conformations in rigor.

Actin in cochlear outer hair cells: A structural basis for cell shape and motility

Threshold sensitivity and frequency selectivity in the cochlea depend on intact outer hair cells and models to explain these phenomena suggest that active mechanical processes, residing in outer hair cells, alter micromechanical properties of the basilar membrane. Auditory sensory hair cells isolated from the inner ear change their shape in response to stimulation in vitro. Three areas of the cell have been suggested to be involved in this form of cell motility: (1) the infracuticular network of actin filaments that is seen as a central core; (2) the cortical layer of actin filaments along the lateral wall; and (3) actin in the outer hair cell cytoplasm. The actin filaments in the stereocilia, the cuticular plate, and the infracuticular network are not involved, since mechanically disrupted cells lacking these structures still shorten. Actin filaments along the lateral wall are not involved in shortening since outer hair cell membrane ghosts do not shorten, however, they retain the ability to constrict so...

Paracrystals of myosin rod.

To help understand the packing of myosin tails in the backbone of the vertebrate striated muscle thick filament, paracrystals of myosin rod, a proteolytic fragment corresponding to the whole myosin tail, have been examined by electron microscopy and image analysis. Two types of paracrystal were observed. Type I paracrystals were similar to those seen by Moos et al. (1975; J. molec. Biol. 97, 1-9). These showed a 14-nm axial repeat, but yielded no other structural information. Type II paracrystals were long, flexible ribbons with more regularity. When negatively stained they exhibited a weak 43-nm axial striation and appeared to be composed of a layer of narrow filaments. Optical diffraction showed that the paracrystals had a rectangular unit cell of dimensions 43 nm axially and 12.4 nm laterally. Transverse sections indicated a paracrystal depth similar to the lateral dimension of the unit cell. Each unit cell contained two filaments arranged antiparallel and related by a two-fold screw axis perpendicular to the length, and in the plane of the ribbon. The filaments probably consist of parallel rod molecules related by axial displacements of 43 nm and higher multiples of 43 nm. The nature of these paracrystals indicates that the myosin tail alone can form structures like thick filament subfilaments. Their structure, based on distinguishable parallel and antiparallel rod interactions, was sensitive to pH and divalent cations in a similar way to the ionic effects on the structure of thick filaments. This behaviour suggests that some of the interactions present in the paracrystal are the same as those in the thick filament.

Effect of alkali treatment on structure of surface layers of elementary polyethylene terephthalate filaments

The change in supermolecular structure of polyethylene terephthalate as a result of hydrolytic degradation of macromolecules in hydrolysis of yarns from Lavsan yarns has been studied by the method of internal reflection IR spectrophotometry. The nonlinear decrease in degree of orientation of macromolecules thereupon has been connected up with a nonlinear decrease in fabric stiffness caused by etching of the surface layer of elementary filaments.

In vitro formation of the “S” layer, a unique component of the fertilization envelope in Xenopus laevis eggs

The extracellular matrix (ECM) of unfertilized Xenopus laevis eggs consists of an elaborate filamentous network in the perivitelline space (PS) and a thick fibrillar vitelline envelope (VE), with a thin layer of horizontal filaments (HF) separating the two. At fertilization this ECM is converted into the fertilization envelope (comprised of the fertilization (F) layer and altered VE), and a third layer, the smooth (S) layer, is formed at the upper boundary of the PS ( Larabell and Chandler, 1988). In this report, we use quick-freeze, deep-etch, rotary-shadow electron microscopy to show that an intact S layer can be formed in vitro by incubation of unfertilized eggs in an exudate obtained from cortical granules. Within 5 min numerous 36-nm-diameter particles assemble in a highly ordered array at the microvillar tips. These particles appear to “melt” and to form patches of smooth material and within 10 min one continuous sheet has formed. The presence of the VE is required for formation of the S layer, and we suggest that the HF layer is the site of assembly.

Topology of the vorticity field in three-dimensional shear layers and wakes

An experimental and numerical study of the three-dimensional transition of plane wakes and shear layers behind a flat plate is presented. Flow visualization techniques are used to monitor the response of laminar flows at moderate Reynolds numbers (≈100) to perturbations periodically distributed along the span. In this way, the formation and evolution of streamwise vortex tubes and their interaction with the spanwise vortices are analyzed. The flow was studied numerically by means of three-dimensional inviscid vortex dynamics. Assuming periodicity in the spanwise and the streamwise direction, we discretize the vorticity field into two layers of vortex filaments with finite core diameter. Comparison between experiment and visualization indicates that important features of the three-dimensional evolution can be reproduced by inviscid vortex dynamics. Vortex stretching in the strain field of the spanwise rollers appears to be the primary mechanism for the three-dimensional transition in this type of flows.

Characterization of the Accessory Layer of the Cuticle of Muscle Larvae of Trichinella spiralis

The accessory layer of the cuticle of infective larvae of Trichinella spiralis has been studied with electron microscopy using cytochemical techniques and chemical extractions. The accessory layer lacks negative charges and carbohydrates demonstrable in vivo. Staining with ruthenium red and tannic acid is interpreted as being consistent with their reactions with phospholipids. Freeze fractures demonstrate an external layer of granules that can be partially released by means of detergents (CTAB and SDS). The granules are considered to be proteins. Their removal makes the worms acid sensitive and prevents them from infecting mice. Extraction of whole worms with ethanol, acetone and methanol (via reaction with 2,2-DMP), or chloroform and methanol destroys an internal layer of filaments. Thin-layer chromatography of chloroform/methanol extracts showed principally ethanolamine phospholipids from the surface of the worms. A model is presented for the molecular organization of the accessory layer. Ethanolamine phospholipids are suggested to occur as tubular micelles. Proteins may attach to these by lipophilic moieties and perhaps by a cryptic sugar group (demonstrated by others) that may penetrate into the hydrophilic core of the lipid micelles.

An experiment on interstrand coupling currents in soldered cables

The effective frequency-dependent transverse resistivity experienced by coupling currents in superconducting cables was studied experimentally. The results extend the theory regarding the effective transverse resistivity of soldered strands to the whole AC domain. An accompanying magnetic effect due to the twist of the filaments has been measured and qualitatively explained by a simple model of the filament layer in the strands. It is seen that the coupling current experiences maximum resistivity if the strands are almost untwisted. >

Intrahepatic biliary tract — oral contraceptives: Electron microscopic examinations of 27 surgical liver biopsies

An electron microscopic study of 27 liver biopsies of patients on oral contraceptives is reported with special attention to the alterations of intrahepatic biliary tract. Bile ductules were normal. Bile canaliculi were damaged in each case with severe alterations of the canalicular membranes. An accumulation of granular, filamentous, lipoid material or collagen fibres could be observed in the lumina of canaliculi. Wide layer of dense actin-like filaments was present in the pericanalicular area and along the lateral cell membrane of liver cells. It is presumed that the ultrastructural alterations of canalicular cell membranes and of pericanalicular cytoplasm are connected with an impaired bile secretion in patients on oral contraceptives.