Location of the Fracture Faces Within the Cell Envelope of Acinetobacter Species Strain MJT/F5/5

The cell wall of the gram-negative bacterium Acinetobacter species strain MJT/F5/5 shows in thin section an external “additional” layer, an outer membrane, an intermediate layer, and a dense layer. Negatively stained preparations showed that the additional layer is composed of hexagonally arranged subunits. In glycerol-treated preparations, freeze-etching revealed that the cell walls consist of four layers, with the main plane of fracture between layers cw 2 and cw 3. The surface of [Formula: see text] 2 consisted of densely packed particles, whereas [Formula: see text] 3 appeared to be fibrillar. In cell envelopes treated with lysozyme by various methods, the removal of the dense layer has detached the outer membrane and additional layer from the underlying layers, as shown in thin sections. When freeze-etched in the absence of glycerol, these detached outer membranes with additional layers fractured to reveal both the faces [Formula: see text] 2 and [Formula: see text] 3 with their characteristic surface structures, and, in addition, both the external and internal etched surfaces were revealed. This experiment provided conclusive evidence that the main fracture plane in the cell wall lies within the interior of the outer membrane. This and other evidence showed that the corresponding layers in thin sections and freeze-etched preparations are: the additional layer, cw 1; the outer membrane, cw (2 + 3); and the intermediate and dense layers together from cw 4. Because of similarities in structure between this Acinetobacter and other gram-negative bacteria, it seemed probable that the interior of the outer membrane is the plane most liable to fracture in the cell walls of most gram-negative bacteria.     

Adhesion-reduced mutants and the wild-typeNectria haematococca: An ultrastructural comparison of the macroconidial walls

We examined the macroconidial wall layers of various strains ofNectria haematococca prior to germ tube emergence. Using freeze-substituted cells, the wall ultrastructure of an adhesion-competent wild-type strain was compared with two adhesion-reduced mutants, LE1 and LE2. At 0 h, the freshly harvested macroconidia of all strains had a similar, bilayered wall and were all nonadhesive. After 1 h, wild-type macroconidia were adhesive and their cell walls exhibited two additional layers not present at 0 h: a pellicular third layer and a thick, outermost fourth layer. Material from the fourth layer was apparently discharged into the surrounding medium. In contrast to the wild type, the mutants after 1 h were adhesion-deficient; the outermost wall layers of LE1 and LE2 differed from each other and from the wild type. There were also differences in the wall layers and extracellular matrices between the mutants and the wild type after 3- and 5-h incubations. Plasma membrane invaginations were not observed at 0 h, but were detected in both wild type and mutant macroconidia at 1, 3, and 5 h. The data demonstrate that in macroconidia ofN. haematococca (1) the wall and associated extracellular matrix undergo major morphological changes prior to germ tube emergence and (2) development of adhesiveness is correlated with the appearance of new wall layers.     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : III. The Surface Layers of Chondrococcus columnaris

An electron microscope study of the myxobacterium Chondrococcus columnaris has revealed the following structures in the peripheral layers of the cells: (1) a plasma membrane, (2) a single dense layer (probably the mucopeptide component of the cell wall), (3) peripheral fibrils, (4) an outer membrane, and (5) a material coating the surfaces of the cells which could be stained with the dye ruthenium red.The ruthenium red-positive material is probably an acid mucopolysaccharide and may be involved in the adhesive properties of the cells. The outer membrane and plasma membrane both have the appearance of unit membranes: an electron-translucent layer sandwiched between two electron-opaque layers. The peripheral fibrils span the gap between the outer membrane and the mucopeptide layer, a distance of about 100 A, and run parallel to each other along the length of the cell. The fibrils appear to be continuous across the ends of the cells. The location of these fibrillar structures suggests that they may play a role in the gliding motility of these bacteria.     

Small Angle X-Ray Scattering of the Thylakoid Membranes of Rhodopseudomonas spheroides in Aqueous Suspensions

The diffraction patterns of particles which have the shape of hollow spheres, i.e. vesicles, can be satisfactorily analyzed by means of a new formula of Weick (1974). This formula is used for the small angle X-ray scattering analysis of aqueous suspensions of thylakoids of Rhodopseudomonas spheroides. Some essential results are: (a) The membrane has a rather asymmetric structure with one layer of low electron density at its inner side and two layers of high electron density near the outer surface of the thylakoids. (b) The distance of the electron density maxima of the latter two layers is 45 ± 5 Å. (c) Between the two maxima is a region of an electron density nearly equal to that of water. (d) The sequence of the peaks is - + 0 + with increasing radius. The peaks extend over an interval of 120 ± 10 Å. (e) The thylakoids are strikingly of the same size. Their diameters, if defined by the outmost layer, vary statistically by about 4% and have an average value of approximately 640 Å.     

Ultrastructure of the secondary tympanic membrane in the human fetus

The normal secondary tympanic membrane in human fetuses was examined by transmission electron microscopy. The membranes in 5- to 9-month-old fetuses consist of the following three layers: (1) an outer squamous epithelial layer facing the middle ear, which is not formed until 4 months old; (2) a middle fibrous layer containing collagen, elastin, fibroblasts and fibrocytes, and (3) an inner layer of flat cells facing the scala tympani. Following the maturation of the fetus the epithelium is getting thinner and fibroblasts are reduced in number, but fibrocytes are increased and collagen and elastin grow gradually in density. The ultrastructure of the secondary tympanic membrane at 8 month is mature in type and shows the same characteristics as in the adult. This membrane has an important and complicated physiological function. The epithelium of the outer layer, with tight junctions and multiple desmosomes, provides a barrier to keep harmful substances out. The stability of the membrane provides protection against rupture, while the elasticity plays a role in the physiology of hearing as well.     

THE POTENTIOMETRIC ANALYSIS OF MEMBRANE STRUCTURE AND ITS APPLICATION TO LIVING ANIMAL MEMBRANES

1. The electromotive forces which arise, if two electrolyte solutions are separated from each other by a layer of any kind, are discussed. A general equation is derived comprising the known equations for diffusion, partition, and membrane (Donnan) potentials as special cases. 2. A method is proposed to analyse membranes potentiometrically with respect to their cation or anion selectivity, their dissolving power for ions, and their influence on ion mobility (migration velocity). 3. The possibility of analysing a membrane composed of several layers of different permeability is discussed. 4. The investigation of the skin of the belly of Rana temporaria leads to the following results. It is composed of at least four layers of different permeability, one of which is specifically permeable to H ions and is very likely identical with the "basal membrane" situated between the stratum germinativum and the corium. The major part of the resting potential of the skin is located across this membrane and is due to the difference of H⁺ concentrations on both sides of the membrane. 5. Experiments on muscle show that the sarcolemma is specifically permeable to H ions. The injury potential of the muscle is attributed to the difference of H⁺ concentration inside and outside the fibre.     

Diffusion Models for the Squid Axon Schwann Cell Layer

The Schwann cell, basement membrane, and connective tissue layers that surround the squid giant axon and constitute barriers to diffusion, were modeled in a number of ways to analyze various experimental results. The experiments considered are (a) the time-course of the potassium concentration in the space between the Schwann cell and the axon membrane (from now on referred to as the F-H space) after an initial loading, (b) the time-course of sodium concentration in the F-H space after a sudden change in the sodium concentration in the external fluid; (c) the time-course of the concentration of tetrodotoxin (TTX) or saxitoxin (STX) in the F-H space after a sudden change in external concentration, including (or not) the effects of specific binding of TTX or STX to sites on the axon membrane and nonsaturable binding to sites in the F-H space or in the spaces (clefts) between Schwann cells; (d) the effects of the F-H space, clefts, and diffusion into the clefts from the outside (from now on referred to as convergence into the clefts) on the measured series resistance.

The analysis shows that (1) in no case is it necessary to include the effects of the convergence into the clefts from the outside; (2) in case a, the basement membrane, connective tissue layers, and the unstirred layer may be neglected, i.e., the clefts are rate limiting; (3) in case b the clefts may be neglected, i.e., the unstirred layer is rate limiting; (4) in most cases the clefts may be replaced by an equivalent thin diffusion barrier.

     

STUDIES ON THE FINE STRUCTURE OF ULTRACENTRIFUGED SPINAL GANGLION CELLS

The following structures were observed in electron micrographs of the mouse spinal ganglion cells: Nissl bodies composed of both aggregated rough-type, largely oriented, membranes of the endoplasmic reticulum and discrete particles; short rodlike mitochondria with well-developed transverse, obliquely or longitudinally arranged cristae, and a relatively typical Golgi complex. The components of ultracentrifuged ganglion cells (400,000 times gravity for 20 minutes) are stratified, the layers appearing in the order of their decreasing density as follows: (1) A microsomal or ergastoplasmic layer which may be further divided into three sublayers without sharp boundaries, namely, a discrete particle layer, a layer of discrete particles and highly distorted membranes of the endoplasmic reticulum, and a layer composed of relatively intact, but stretched membranes of the endoplasmic reticulum and discrete particles. (2) Mitochondria constitute a relatively broad layer. They are sometimes stretched; however, they retain most of their fine structure. The stratified nucleus is found within the mitochondrial layer. (3) A relatively wide layer of tightly packed vesicles. (4) At the centripetal end, resting against the cell membrane, are a few lipid vacuoles. A comparison is made between the ultrastructure of the stratified layers in situ and those described by others in differentially ultracentrifuged homogenates.     

STUDIES ON BLOOD CAPILLARIES : I. General Organization of Blood Capillaries in Muscle

The wall of the blood capillaries of skeletal muscles (diaphragm, tongue, hind legs) and myocardium of the rat, guinea pig, and hamster consists of three consecutive layers or tunics: the endothelium (inner layer), the basement membrane with its associated pericytes (middle layer), and the adventitia (outer layer). The flattened cells of the endothelium have a characteristic, large population of cytoplasmic vesicles which, within the attenuated periphery of the cells, may attain a maximum frequency of 120/µ² of cell front and occupy ~18% of the cytoplasmic volume; these values decrease as the cells thicken toward the perikaryon. The vesicles are 650–750 A in over-all diameter and are bounded by typical unit membranes. They occur as single units or are fused to form short chains of two to three vesicles. Each configuration may lie entirely within the cytoplasm or open onto the cell surface. In the latter case, the unit membrane of the vesicle is continuous, layer by layer, with the plasmalemma. Chains of vesicles opening simultaneously on both the blood and tissue fronts of the endothelial tunic have not been observed either in sections or in a tridimensional reconstruction of a sector of endothelial cell cytoplasm. Adjacent endothelial cells are closely apposed to one another and appear to be joined over a large part of their margins, possibly over their entire perimeter, by narrow belts of membrane fusion (zonulae occludentes). Except for tongue capillaries, patent intercellular gaps are rare or absent. The middle layer is formed by a continuous basement membrane (~500 A thick) and by pericytes which lie in between leaflets of this membrane. The tips of the pericyte pseudopodia penetrate through the inner leaflet of the basement membrane and join the endothelium in maculae occludentes. The adventitia is a discontinuous layer comprising cellular (macrophages, fibroblasts, mast cells) and extracellular (fibrils, amorphous matrix) elements. The same general type of construction appears to be used along the entire length of the capillary.     

The soft tissue of Jeholopterus (Pterosauria,Anurognathidae, Batrachognathinae) and the structure of the pterosaur wing membrane

The soft tissue preserved in the holotype (IVPP V12705) of Jeholopterus ningchengensis from the Daohugou Bed (Late Jurassic or Early Cretaceous) of China is described in detail. The plagiopatagium can be divided into the distal, comparatively more rigid actinopagatium and a proximal, more tensile tenopatagium. The actinopatagium extends from the wing finger to the articulation between the humerus and the forearm, and shows the presence of at least three layers containing actinofibrils. In each layer, the actinofibrils are parallel to subparallel, but this direction diverges from layer to layer. When distinct layers of actinofibrils are superimposed (owing to taphonomic compression), a reticular pattern is generated. The presence of layers with differently oriented actinofibrils is widespread in this pterosaur. A well-developed integumental covering formed by fibres (here named pycnofibres) that are thicker than the actinofibrils is present. Ungual sheaths that extend the length of the pedal and manual claws of this taxon are also observed. Although the understanding of the mechanical properties of the wing membrane is hampered by the lack of knowledge regarding the composition of the actinofibrils, the configuration observed in Jeholopterus might have allowed subtle changes in the membrane tension during flight, resulting in more control of flight movements and the organization of the wing membrane when the animal was at rest.     

Pollen development of Cardiocrinum giganteum (Wall.) Makina in China

In this article, we studied the pollen morphology and wall development, microsporogenesis, male gametophyte development, and anther wall structure changes during pollen development of Cardiocrinum giganteum (Wall.) Makina from the genus Cardiocrinum (Endl.) Lindl. (Liliaceae) using paraffin sections, scanning and transmission electron microscopy, and fluorescence microscopy. The results showed that C. giganteum has oval-shaped pollen with a single sulcus and reticulate ornamentation. The exine is of the semi-tectum type and can be divided into the tectum layer, columellate layer and basal layer. Meiosis in the microsporocyte is accompanied by successive cytokinesis. The mature pollen is three-celled. The anther wall prior to maturity is built by one layer of epidermis, 1–2 layers of endothecium cells, 4–5 middle layers and 2 layers of tapetum, while upon maturity it is only built by one layer of epidermis, one layer of endothecium cells and one middle layer. The tapetal cells are secretory, with two or more nuclei. Ubisch bodies originate from rough endoplasmic reticulum except a few from mitochondria.     

The structure and associations of the double S layer on the cell wall of Aquaspirillum sinuosum

Aquaspirillum sinuosum cell walls bear two paracrystalline, proteinaceous surface layers (S layers). Each shows a different symmetry: the inner layer is closely apposed to the outer membrane and is a tetragonal array (90 degrees axes; 5-nm units; repeat frequency 8 nm); the outer layer is a hexagonal array on the external surface (14-nm units; repeat frequency 18 nm) and, although the units have a six-pointed stellate form, the linkage between units is not resolved. The outer layer consists of a major 130-kDa protein and a 180-kDa minor component; these co-extract, co-assemble, and are inseparable by hydroxylapatite chromatography or by recrystallization. The solubilizing effects of reagents suggest stabilization by hydrogen bonding and Ca2+. The two outer layer proteins are serologically related and show partial identity by peptide mapping. Periodic acid--Schiff staining of the 180-kDa band suggests that this may be a glycosylated form of the 130-kDa component. The inner layer components form a doublet of 75- and 80-kDa polypeptides with extreme resistance to extraction. Close apposition to the outer membrane, resistance to chaotropes, aqueous insolubility, and behaviour in charge-shift electrophoresis suggest hydrophobic interaction between subunits and an integral association with the outer membrane.     

The ultrastructural organization of the basement membrane of Bowman's capsule in the rat renal corpuscle

Summary The basement membrane of Bowman's capsule (BCBM) of the rat was studied by means of a modified tissue-preservation technique for transmission electron microscopy, which avoids the usual thorough fixation in OsO₄ and applies tannic acid and uranyl acetate for staining (Sakai et al. 1986). At most sites the BCBM is multilayered, consisting of one to seven dense layers separated by electron-lucent layers. The latter, which can be termed laminae rarae, contain fine filaments which connect the dense layers to each other and the innermost dense layer to the basal cell membrane of the parietal epithelium. The laminae densae are basically composed of fine filaments arranged in an anastomosing pattern. Individual filaments ranging from 5 to 15 nm in diameter, combine to form filament bundles up to 100 nm in thickness and 1 to 2 m in length. Within a dense layer, filaments and filamentous bundles are oriented mainly in the same direction. Often the inner dense layers do not form a continuous sheet, and the filamentous bundles are arranged in anastomosing or spiral patterns to form a ribbon-like structure that we call a microligament. These microligaments are often embedded in basal furrows of the parietal epithelium and are best developed around the vascular pole. Intracellular actin bundles of the parietal cells are regularly associated with these extracellular ribbon-like structures of the basement membrane. In conclusion, the BCBM has an unusual structure: the laminae densae are characterized by their filamentous nature and are arranged in different patterns, i.e. as a multilayered mat and as microligaments.Fellow of the Deutscher Akademischer Austauschdienst     

Ultrastructure and resistance to water loss in eggs of Acanthoscelides obtectus Say (Coleoptera: Bruchidae)

The mature oöcyte of Acanthoscelides obtectus is surrounded by three envelopes: an external layer, a chorion and a vitelline membrane. The external layer is secreted by the walls of the lateral oviducts. The chorion and vitelline membrane are secreted by the follicular cells. The vitelline membrane becomes very compact during the hour following fertilization and laying. The chorion is composed of three layers, one of which has a paracrystalline ultrastructure.Mature, unfertilized, chorion-containing oöcytes, whose vitelline membranes are loose, dehydrate rapidly in a dry atmosphere after laying or after removal from the lateral oviducts. Fertilized eggs are quite resistant to desiccation: after 12 days at 25°C and 5% relative humidity, viable larvae are obtained.The compact vitelline membrane is the most effective protection against dehydration. The chorion and the external layer are much less effective in preventing water loss from the egg.The retention of eggs in the lateral oviducts does not seem to lead to any modification of the structure of their envelopes.     

Ultrastructure and development of the extensible abdominal intersegmental membranes of the female migratory locust

The cuticle of the retracted extensible membrane of the female locust is 300 mum thick and below its highly folded epicuticle there is a zone (5 mum thick) of helicoidally oriented laminae of microfibrils (lamellae), an elastomer layer (180 mum thick) of microfibrils with no preferred orientation and a subcuticular zone (1 mum thick). The epidermal cell layer has an extensive system of junctional specializations and pore canals traverse the cuticle to the helicoidally oriented lamellae. In the newly ecdysed adult the elastomer layer is absent and the helicoidally oriented lamellae are incomplete. Essentially the membrane consists of an elastomer layer contained between two stable layers cross-linked by pore canals, one to the other. When in the plasticized state the membrane combines low stiffness with high extensibility and during extension the elastomer layer flows. Recovery is effected by muscles and when the two stable layers have returned to their unstretched states the fluid elastomer is again evenly distributed. There is an increase in the water content and in the volume of the cuticle when it is fully extended. The ultrastructure of the extensible membrane is compared with those of the inextensible membranes from male and female locusts.     

Oogenesis of Cardiochiles nigriceps viereck (Hymenoptera : Braconidae): Histochemistry and development of the chorion with special reference to the fibrous layer

A fibrous layer on the surface of eggs of the parasitoid, Cardiochiles nigriceps (Hymenoptera : Braconidae), has been implicated by earlier studies in the evasion from encapsulation by host hemocytes. The present histochemical and ultrastructural study was undertaken to characterize fibrous layer material and to determine the source of fibrous layer and other components of the eggshell. The fibrous layer contains neutral glyco- or mucoprotein; acidic mucoproteins or glycosaminoglycans are absent. The mature eggshell is resolved into 5 morphologically distinct layers by electron microscopy: (from inner to outer) vitelline envelope, endochorion, an electron-dense “irregular layer”, papilliary layer and fibrous layer. During oogenesis each eggshell layer is laid down sequentially in the order mentioned above. Eggshell material appears to be produced by the follicle cells because these develop extensive rough endoplasmic reticulum and golgi apparatus and exhibit apparent exocytotic activity at the plasma membrane adjacent to the egg.     

THE ULTRASTRUCTURE OF MOUSE LUNG : SOME REMARKS REGARDING THE FINE STRUCTURE OF THE ALVEOLAR BASEMENT MEMBRANE

The fine structure of the alveolar basement membrane of mouse lung was discussed on the basis of three electron micrographs. The basement membrane, i.e., the intercellular layer between endothelium and alveolar epithelium, was found to be of variable width. In its thin parts it appeared rather homogeneous, and did not reveal well defined layers of fibrils. In its thicker portions, some of which may be due to oblique sectioning, cell fragments could be seen lying inside the basement membrane layer. Their exact nature was not determined. In other thickened portions of the membrane bundles of slender (about 23 to 25 mµ) fibrils were found and were tentatively interpreted as collagen fibrils, in spite of the fact that a periodicity could not be observed.     

The interaction of fetuin with phosphatidylcholine monolayers. Characterization of a lipoprotein membrane system suitable for the attachment of myxoviruses

1. An artificial membrane system was formed by spreading at air/water and oil/water interfaces, by using phosphatidylcholine and the glycoprotein fetuin (mol.wt. 48400). 2. The plot of increase of interfacial pressure against amount of protein added beneath a monomolecular film of phosphatidylcholine showed two discontinuities, corresponding to the completion of two distinct layers of protein: (a) largely denatured and closely associated with the polar head groups of phosphatidylcholine, possibly with penetration of non-polar protein groups between the phosphatidylcholine molecules and (b) an additional adsorbed layer of substantially native fetuin in either a close-packed or open-lattice array. A more compactly organized membrane was apparently formed at pH7.4 with 1mm-Mg²⁺ in the aqueous phase than without Mg²⁺; at 15mm-Mg²⁺, more random adsorption of protein appeared to take place. Qualitatively similar results were obtained at pH5.1 with 1mm-Mg²⁺. Closer initial packing of the phosphatidylcholine layer decreased both the magnitude of the interfacial pressure change and the amounts of protein bound in the two layers. 3. The amount of N-acetylneuraminic acid released by neuraminidase (EC 3.2.1.18) in the subphase was measured at pH5.1; a mean distribution of 9.7×10¹³ residues/cm² was calculated for the completed second protein layer.     

Conditional Ablation of the Choroideremia Gene Causes Age-Related Changes in Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm^Flox, Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch’s membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5–6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm^Flox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.     

Fine structure and morphogenesis of the micropylar apparatus in the medfly Ceratitis capitata (Wiedemann) (Diptera : Tephritidae)

The micropylar apparatus (MA) in Ceratitis capitata (Diptera : Tephritidae) is a cone-like protrusion, 18 μm long, at the anterior pole of the egg, and exhibits about 40 follicle cell imprints externally. It consists of chorionic and vitelline membrane parts. The first contains at least a 3 μm wide micropylar canal; the tip of the MA is covered by a “tuft” and includes the micropyle, i.e. the entrance of the micropylar canal. The canal leads to the vitelline membrane part, where it forms a pocket. The sperm enters the oocyte by passing through the micropyle-micropylar canal-pocket route.At least 40 follicle cells participate in the formation of the micropylar apparatus. Two of these form 2 projections, which are tightly connected, and serve as a template for the formation of the canal and the pocket. Throughout their length, both projections have microtubules in parallel arrangement. During oogenesis, the remaining micropylar cells secrete the successive eggshell layers, i.e. the vitelline membrane, the wax layer, the innermost chorionic layer, the endochorion, and the exochorion. Towards the end of oogenesis, the 2 projections degenerate, and the canal becomes available for sperm passage.