Membrane Strands Research Articles

Neurobiological Studies of Experimental Diphenylhydantoin Intoxication

The effect of chronic administration of diphenylhydantoin on the fine structures of the rat cerebellum was ultrastructurally investigated. Materials from nine control rats and 16 experimental rats to which 200 to 300 mg/kg b.wt. of diphenylhydantoin was administered orally for 12 months were studied with the light and electron microscopes. The most striking changes were observed in the presynaptic boutons located predominantly in the molecular layer and the neuropil of deep cerebellar nuclei and sometimes in the granule cell layer. The changes consisted of the proliferation of either interconnected tubules 150 to 350 A in diameter associated with membranous scrolls and/or membranous strands separated by a cleft-like space, or parallel tubules about 600 A in diameter with a honeycomb-like architecture in the cross section, and hypertrophy of the boutons. Other alterations were a generalized increase in the density of the axoplasmic matrix, the accumulation of mitochondria, vesicular structures and dense bodies, an increased number of neurofilaments and a decreased number of synaptic vesicles. The axon displayed morphological abnormalities similar to those found in the terminal boutons. Along with the synaptic and axonic changes mentioned above, the perikaryal alterations in the Purkinje cell and in granule cell layers and deep cerebellar nuclei were also found, but they were generally not so prominent so that they were considered to reflect a non-specific reaction to synapto-axonic changes rather than a primary degenerative change. However, in contrast to the neuronal changes, no indication of damage of the glial elements was found, nor were the vascular changes.

Electron microscopy of the red pulp of the dog spleen including vascular arrangements, periarterial macrophage sheaths (ellipsoids), and the contractile, innervated reticular meshwork.

The vascular and stromal arrangements of the red pulp in congested and contracted dog spleens were studied by transmission electron microscopy. Each dog had been injected intravenously with Thorotrast to label actively endocytizing cells. Only macrophages ingested Thorotrast. The proximal portion of each arterial capillary was surrounded by a "periarterial macrophage sheath" (PAMS), a term we introduce to replace the term "ellipsoid". PAMS were composed of a fine meshwork of reticular cells and reticular fibers which held tightly-packed macrophages and interspersed blood cells. These macrophages, as well as those in the reticular meshwork of red pulp, contained Thorotrast, cell debris, and deposits of hemosiderin. The arterial capillary at the center of each PAMS was formed by parallel, rod-shaped endothelial cells and discontinuous layers of basement membrane and reticular-cell cytoplasm. PAMS were tapered at their distal ends; the terminal portion of the arterial capillary continued beyond the PAMS to end in the reticular meshwork of red pulp. Endothelial cells in the terminal arterial capillaries were separated by gaps through which blood cells passed into the spaces of the reticular meshwork of red pulp. The reticular meshwork was formed by reticular cells which appeared to be specialized for contraction. These cells were filled with thin filaments and possessed plasmalemmal dense bodies as found in smooth muscle cells. Furthermore, the reticular meshwork was innervated by unmyelinated adrenergic axons which probably were derived from nerves that followed arterioles. Axons were enclosed in surface invaginations of cells which were similar to reticular cells in shape and cytologic detail and which we called "axon-bearing reticular cells". Axon-bearing reticular cells were inserted between the branches of the reticular cells that formed the meshwork. Venous sinuses formed an anastomosing system of vessels draining into pulp veins which then joined trabecular veins. Sinuses were formed by parallel, rod-shaped endothelial cells encircled by strands of basement membrane and reticular-cell branches. Endothelial cells lay closely side by side except where interendothelial slits were opened by blood cells passing into the lumen or by pseudopodia of macrophages which lay outside the sinus. Cell traffic across the sinus wall was greatest in areas where blood cells were mixed with plasma. Congested spleens stored concentrated red cells in both sinuses and the reticular meshwork; contracted spleens were emptied of blood. The reticular meshwork may contract to assist trabecular and capsular smooth muscle in expelling stored red cells and effecting hemoconcentration.

Experimental modulation of occluding junctions in a cultured transporting epithelium.

The experimental opening and resealing of occluding junctions in monolayers of cultured MDCK cells (epithelioid of renal origin) was explored by measuring changes in the electrical resistance across the monolayer and by freeze-fracture electron microscopy. As in natural epithelia, the function of occluding junctions as permeability barriers specifically depends on extracellular Ca++ concentration and fails if this ion is replaced by Mg++ or Ba++. The removal of Ca++ and the addition of EGTA to the bathing medium opened the junctions and reduced the transepithelial resistance. Resealing was achieved within 10-15 min by restoring Ca++. Quantitative freeze-fracture electron microscopy showed that junctional opening, caused by lack of Ca++, was accompanied by simplification of the pattern of the membrane strands of the occluding junction without disassembly or displacement of the junctional components. Resealing of the cellular contacts involved the gradual return to a normal junctional pattern estimated as the average number of strands constituting the junction. The occluding junctions were also opened by the addition of the ionophore A23187, suggesting that the sealing of the contacts requires high Ca++ on the extracellular side and low Ca++ concentration of the cytoplasmic compartment. The opening process could be blocked by low temperature (7.5 degrees C). Resealing did not depend on serum factors and did not require protein synthesis; therefore, it seems to be caused by reassembly of preexisting membrane junctional components. The restoration of the junctions occurred simultaneously with the establishment of ion-selective channels; the Na+/Cl- and the cation/cation selectivity were recovered with the same time-course as the electrical resistance. The role of the cytoskeleton in the process of junctional reassembly is reported in the companion article.

Fine structure of nuclei and cytoplasm of osteoclasts in Paget's disease of bone.

To investigate the fine structural features of nuclei and cytoplasm in osteoclasts from patients with Paget's disease of bone, eight bone biopsies were examined. Unusual paracrystalline filamentous formations were found with the nuclei of both actively resorbing and degenerating osteoclasts. These formations were made up of parallel arranged filaments which, in most cases, clustered together regularly. The filaments were slightly argyrophilic and this helped to differentiate them from the surrounding nuclear matrix, even when they were not aggregated in clusters. Argyrophilic filaments were also found loosely aggregated inside the cytoplasm of osteoclasts. In three biopsies two additional and unusual cytoplasmic inclusions were found. The first consisted of irregularly branching, highly argyrophilic membranous strands which were located inside membrane-bound vacuoles. The second consisted of spindle-shaped structures enclosed by two membranes and containing three or more tubules. It is suggested that these results stand as further morphological evidence in favour of the view that a virus, possibly of the paramyxovirus class, has a pathogenetic role in Paget's disease of bone.

Operation einer kongenitalen Katarakt bei einem Sibirischen Tiger

A cataract operation in a young Siberian tiger is reported. This 5-month-old tiger, presenting with blindness, showed bilateral residual pupillary membranes and anterior polar cataracts combined with nuclear cataracts. After unilateral intracapsular extraction of the lens, the tiger adapted well to the improvement in sight and behaved almost like an animal with normal vision. The animal died 3 months post-operatively from unknown cause. Histological examination of the aphakic eye showed, at the site of operation on the cornea, an intact epithelium with discrete infiltration of the stroma together with low-grade vascularisation. In the eye not treated operatively, both an anterior polar cataract with adherent pupillary membrane strands and a central cataract were seen. Of particular note, when comparing this large wild cat with the domestic cat, was the marked development of the tapetum lucidum (up to 30 cell layers).

Preparation of plasma membranes from isolated cells of newborn rat brain

A procedure is presented for the isolation of plasma membranes from a suspension of immature brain cells. The cells were homogenized in a hypotonic medium and plasma membranes and other subcellular fractions were separated by differential centrifugation in sucrose. The recovery of protein in the plasma membrane fraction was 1.3%. 1. 1. Electron microscopy showed that plasma membranes were long, mainly open membrane strands. No nerve endings, myelin or mitochondria could be detected in the fraction. 2. 2. The concentrations of protein, phospholipid and RNA were recorded in the subcellular fractions. Phospholipid was concentrated in plasma membranes 2.5 times above the level in the homogenate. By contrast, the RNA content in the fraction was low. 3. 3. The activities of (Na +−K +)-activated ATPase, K +-stimulated phosphatase and 5′-nucleotidase were concentrated 7.1, 3.1 and 3.6 times, respectively, in the plasma membrane fraction compared to the activities in the homogenate. These results suggest that even in the brain (Na +−k +)-activated ATPase is mainly localized in the plasma membrane.

Characterization of proteins and glycoproteins of surface membranes isolated from immature brain cells

Surface membranes were isolated from a suspension of immature neurons. Electron microscopy revealed long membrane strands and vesicles. Proteins of surface membranes, separated by SDS polyacrylamide gel electrophoresis, contained three major subunits with molecular weights ranging from 45 000 to 60 000. Staining gels for carbohydrates exposed several glycoprotein bands with high molecular weights. Incorporation of [ 3H] leucine and [ 3H] fucose in surface membranes was much lower than incorporation in the homogenate. Labelled leucine and fucose entered surface membrane even after a chase indicating a continuous transport.

A rapid, simple method for isolating pinocytotic vesicles and plasma membrane of lung

We have found means of isolating pinocytotic vesicles and attached plasma membrane from the low speed (200 × g) supernatant of homogenates of lung. In lung, 5′-nucleotidase is restricted to pinocytotic vesicles and areas of incipient vesicle formation along the plasma membrane. In our method, P i released from AMP is precipitated as lead phosphate at the subcellular site of 5′-nucleotidase. The resulting increase in density allows collection of pinocytotic vesicles and attached plasma membrane as a pellet after centrifugation through sucrose ( d = 1.18) at 250 × g. The final pellet contains long strands of plasma membrane, and the vesicles retain their characteristic morphology including the delicate diaphragm covering their stomata. The entire procedure can be performed in less than 90 min.

Replication of bacteriophage M13: VI. Attachment of M13 DNA to a fast-sedimenting host cell component

During RF 3 3 Abbreviations used: RF, replicative form; RFI, the covalently closed double-stranded ring form; RFII, the double-stranded ring form containing one or more single-stranded breaks; SS, single strand; TdR, thymine deoxyriboside; dRFI, denatured RFI; PFU, plaque-forming unit. replication both parental and pulse labeled M13 DNA are associated with a fast sedimenting host cell component, tentatively identified as membrane. RF molecules containing parental label have equal amounts of linear and circular parental strands. Most of the pulse label in RF molecules is contained in strands of unit length or shorter. The proportion of pulse-labeled M13 DNA associated with membrane decreases with increasing pulse lengths. The freely sedimenting M13 DNA components are identified as RFI and RFII. Pulse label is found in both strands of membrane bound RFII, but in only the viral strand of freely sedimenting RFII. M13 DNA pulse labeled during single-strand synthesis also appears to be membrane associated, the proportion decreasing with increasing pulse lengths. The pulse label is contained exclusively in the viral strand of the bound RF molecules.

Post-fertilization functions of annulate lamellae in the periphery of the egg of Spirorbis borealis (Daudin) (Serpulidae=Annelida).

Post-fertilization changes in the periphery of the egg of Spirorbis borealis are described. The nuclear envelope and cell membrane are formed by the coalescence of short strands of membrane perhaps produced by the Golgi complex. Annulate lamellae are abundant in the forming cells giving rise to endoplasmic reticulum and apparently contributing to genesis of mitochondria. This possibility is discussed.

Enrichment of 5′-nucleotidase in membrane fragments isolated from acathamoeba sp.

A purified membrane fraction enriched in 5′-nucleotidase by 12–19-fold relative to homogenate on a specific activity basis was isolated from amoeboid cells of Acanthamoeba sp. The purified preparation also showed an enrichment in ATPase activity but membranes possesing glucose-6-phosphatase and succinic dehydrogenase activities were relatively absent. From electron micrographs it was apparent that the isolated fraction was a clean preparation of membranous strands and vesicles. The increased specific activity of 5′-nucleotidase, which has been shown to be a marker enzyme for plasma membrane in several mammalian tissues, strongly suggests that the preparation from Acanthamoeba sp. is purified plasma membrane. Moreover, the enzyme appears to be located primarily on the inner surface of the plasma membrane, since its activity was markedly latent in intact cells.

The organization of the connective tissue of Discocelides langi, (Turbellaria, polycladida)

The organization of the connective tissue of the polyclad turbellarian Discocelides langi has been studied using light microscopical, histochemical and electron microscopical techniques. Filamentous components are found in the subepidermal membrane and in the body, especially around muscle cells. The subepidermal membrane is complex, consisting of an outer limiting component and the membrane proper. The membrane proper is composed of filamentous lamellae. The filaments, probably belonging to the collagen family of proteins, form an orthogonally arranged grid system. Large amounts of ground substance are found in the interior of the body. Histochemical observations indicate that it probably consists of both neutral and acid polysaccharides in combination with protein components. On electron micrographs it presents itself as a finely granular to fibrillar net-like material of low density. A frequently occurring component is a prominent system of membranous strands, composed of polysaccharide-protein materials. They are in continuity with and similar to cellular mucous coats or glycocalyx components. The inherent difficulties in identification of connective tissue cells are discussed. In the peripheral parts of the animal, cells which are closely apposed, and reminiscent of planarian fixed parenchyma cells are found. In the interior parts cells similar to these are present but are embedded in ground substance. The cells often are large and branched and contain a cytoplasm with few organelles and of very low density on electron micrographs. Another type is strongly basophilic due to a high content of RNA. The fine structural morphology is very varied but is always characterized by the presence of a great number of ribosomes, either free or membranebound. This type possibly represents the formative cells responsible for synthesis of protein filaments and ground substance. The connective tissue system found is discussed in relation to other types of turbellarian connective tissue. Also the functional implications are commented on.