Trilaminar Membrane Research Articles

The structure of yellow yolk in the domestic fowl

A microscopical study of sections of the yellow yolk in the eggs and large oocytes of the domestic fowl (Gallus domesticus) showed that the yolk mass consisted of tightly packed spheres. Each sphere was bounded by a trilaminar membrane and contained numerous subdroplets dispersed in a lighter groundwork. The subdroplets were formed, in part, of granules of mean diameter 11.4 nm; the groundwork was formed of particles of mean diameter, 26.7 nm. Lamellar bodies, 100–200 nm in diameter, were present in the groundwork of aged yolk spheres. Using intravenously injected ferritin as a tracer, it was estimated that the yolk spheres in the oocyte periphery were formed within a period of 15 min.

Formation of the plasma membrane during regeneration ofParamecium caudatum

Fragments ofParamecium caudatum cells obtained by merotomy were fixed in 1% OsO4 within 5 seconds after cutting. The ultrastructure of the damaged area of the fragment was studied in oriented ultrathin sections and by scanning electron microscopy. The cytoplasm exposed by merotomy was covered during a few seconds with a new membrane. This was a typical trilaminar membrane continuous with the plasma membrane covering the undamaged surface of the cell. The surface over the wound was wrinkled into irregular grooves and ridges. The cytoplasm, mitochondria and trichocysts in the injured region were electron translucent. The cytoplasm under the new membrane contained an unusually high amount of small membrane vesicles, 20–90 nm in diameter. These were probably the remnants of subpellicular alveoli and the plasma membrane destroyed by microsectioning. The possibility that the exposed cytoplasm would be covered by mere shifting of the existing plasma membrane can be excluded. The complex structure of the cortex with its subpellicular alveoli and regularly distributed cilia provide a strong argument against this notion. It seems probable that the new membrane was built up from the available molecular material,e.g., phospholipids and proteins present in the cytoplasm. Fragments of the membrane and alveolar membranes in the form of small vesicles may have also been included into the new membrane.

Observations on the [formula omitted][formula omitted] formation, development and differentiation of trilaminar vesicles formed from enzymatically dispersed porcine blastocysts

The formation of trophoblastic vesicles and their use as a model for cell-to-cell interactions in the embryonic development of the mouse have been extensively studied both in vitro and in vivo . In this report, we describe the formation of trilaminar vesicles in vitro from enzymatically dispersed porcine blastocysts. Trilaminar vesicles were generated from single cell suspensions of enzymatically dispersed porcine blastocysts recovered 12 through 15 days post-breeding. Blastocysts at 10 and 11 days of development failed to form TV. No preference for self-aggregation was apparent as evident by the formation of vesicles containing cells from two different blastocysts origins. Two distinct morphological types of vesicles were formed: vesicles that displayed complete homogeneity in cell density and vesicles that had a small area of greater cell density resembling the embryonic disc. Some TV displayed localized areas of nucleated eosinophilic cells within the mesenchymal layer of the trilaminar membrane. In rare instances (2%) TV displayed contractility associated with a raised area of cells on the outer surface of the membrane. The cells comprising this contractile region were positive for Ca ++-activated myofibriliary ATPase acitivity. Transfer of TV to the uterine horn of a gilt failed to delay a return to estrus. Transfer of TV to the uterine horn of pseudopregnant rabbits induced a localized inflammation and infiltration of leukocytes three days after transfer but no inflammation was evident at 9 or 16 days post-transfer.

A rickettsiales-like infection in the pacific razor clam, Siliqua patula

An intracellular procaryotic rickettsiales-like organism was discovered in the digestive absorptive epithelium of the Pacific razor clam, Siliqua patula, from the Pacific Coast of Washington State. Of 80 animals examined, 30% contained the organisms, and all positive animals were between 1.5 mm and 3.0 cm shell length. The organisms appeared as basophilic inclusions averaging 5.0 μm in diameter. Ultrastructurally, the inclusions were membrane-bound vacuoles containing procaryotic forms which averaged 0.8 μm by up to 1.8 μm in length. These organisms had a double layer of trilaminar membrane which appeared to form blebs and outer membrane vesicles. The organisms were pleomorphic by both histological and electron microscopical examination. The host response to infection appeared to consist only of the rounding and detachment of the infected cells. Specific immunofluorescent tests for Rickettsia rickettsia, R. prowazekii, Coxiella burnetti, Chlamydia psittaci, and Legionella pneumophila were all negative when tested on impression smears of infected clam digestive gland.

Mast Cell Degranulation in the Evolution of Acute Eruptive Guttate Psoriasis Vulgaris

Clinically normal psoriatic skin (CNPS) and psoriatic lesions (PLs) were studied for mast cell degranulation (MCD) in patients with acute eruptive guttate psoriasis vulgaris (AEGP) following penicillin-treated acute streptococcal throat infection. The clinically manifest duration of psoriasis at the time of the biopsies was 2, 5, 10, 14, or 21 days. Two types of MCD were distinguished. Type I was characteristic for those portions of the CNPS in which vascular and epidermal changes were detected, while the PLs showed both Type I and Type II MCD. In Type I MCD the extruded granules (MCGs) in the immediate vicinity of the mast cells appeared as intact bodies encased in a distinctly trilaminar membrane. Around subepidermal and subpapillary blood vessels, in stratum papillare without proximity of blood vessels, beneath the epidermal-dermal junction, in lamina lucida, and in intercellular space of strata basale and spinosum the MCGs appeared partly as intact structures and partly in more or less disintegrated form. In Type II MCD the MCGs were extruded without perigranular membranes. The data here presented showed that MCD is an early and constant feature in the evolution of AEGP.

Preparation of a human lung purified plasma membrane fraction: confirmation by enzyme markers, electron microscopy, and histamine H1 receptor binding.

A simple and rapid method of isolating plasma membranes from human peripheral lung tissue is described. The method involves homogenization of tissue in 0.25 M sucrose-buffered medium followed by differential and sucrose density gradient centrifugation. Enzymatic and morphological characterization of the plasma membrane fraction revealed minimal contamination by nonplasma membrane fragments. The isolated plasma membranes showed an 18-fold purification of 5'-nucleotidase activity compared to the original homogenate. Electron-microscopic studies of the plasma membrane fraction revealed the presence of small membrane vesicles having a trilaminar membrane structure. To further examine the purity of the plasma membrane preparation, the binding of the H1 receptor antagonist, 3H pyrilamine, to the plasma membrane-enriched fraction was compared to the binding to crude membrane preparations. Both the plasma membrane-enriched fraction and the crude membrane preparation had similar Kd's for the histamine antagonist, but the plasma membrane-enriched fraction had a threefold greater binding capacity, reflecting the relative enrichment of plasma membranes of the preparation. Thus, a method has been developed for the isolation of plasma membranes from human peripheral lung which should provide material for a variety of biochemical and pharmacological studies.

Studies on human gastric mucosal zymogen granules and their zymogens

Intact human gastric mucosal zymogen granules (ZG) were detected in specimens from surgical resections of one patient with gastric adenocarcinoma and two with benign gastric ulcer. Both large ZG with unilaminar membranes and smaller ZG with trilaminar membranes were identified by electron microscopy. The zymogens in the ZG and in mucosal extracts were separated by gel electrophoresis. Slow-moving Protease (SMP) was seen in the whole mucosal extracts but was absent from ZG. One specimen of pyloric mucosa showed a striking absence of ZG. Despite the absence of ZG, pyloric mucosa showed Pepsinogens 2–5 (constituents of PG I) as well as Pepsinogens 6 and 7 (constituents of PG II) and SMP.

Tubular intranuclear inclusions in a patient with methotrexate-associated pulmonary fibrosis

A forty-six year old male, who had received methotrexate therapy for the past year for psoriasis of twenty years' duration, presented with a cough which had persisted for two to three months. There was a two year history of dyspnea upon exertion as well as intermittent fever, chills and sweats. The methotrexate treatment did not improve the skin condition and, because there was evidence of hepatic toxicity, the drug was discontinued. Radiological examination of the chest revealed diffuse bilateral interstitial fibrosis with extensive changes; a subseguent lung biopsy was obtained. Light microscopic evaluation demonstrated marked interstitial fibrosis, chronic inflammation, thickening of the alveolar septa, proliferation of alveolar macrophages, occasional fibrin deposits and multinucleated giant cells.Electron microscopic studies revealed massive fibrosis with a mixed population of infiltrating inflammatory cells. Some alveolar spaces were lined by metaplastic cuboidal epithelial cells, and in numerous residual type II pneumocytes, aggregates of branching tubules lined by trilaminar membranes were identified within the nucleus.

Isolation and chemical composition of the cytoplasmic membrane of the archaebacterium Methanospirillum hungatei.

The cytoplasmic membrane of Methanospirillum hungatei was isolated from osmotic lysates of spheroplasts, with yields of 7-8% of the cell dry weight. Cytoplasmic contamination was negligible, as judged by the removal of soluble enzymes. The cytoplasmic membrane consists of lipid (35-37%), primarily as a biphytanyldiglycerol tetraether glycolipid; protein (45-50%); and carbohydrate (10-12%). Ultra-thin sections showed that the trilaminar membrane formed vesicles with a maximum diameter of 0.4 microns. Protrusions of membrane projecting from the vesicles were seen often in negatively stained preparations. Fractionation of M. hungatei cells grown in the presence of [14C]mevalonic acid revealed that 90% of the phytanyl lipids were present in the cytoplasmic membrane band, with two minor bands accounting for the remainder of the label. Approximately 50% of the galactose, glucose, and mannose present in the cytoplasmic membrane was found in lipid extracts, while the remainder of these sugars and 98% of the rhamnose were present as nonlipid sugars. The cell sheath, isolated with a yield of 13% of the cell dry weight, contained the same sugars as the cytoplasmic membrane, but in very different proportions. Amino acid analysis of the membrane proteins showed that hydrophobic amino acid residues made up 37% of the total, neutral amino acids, 39%, basic, 8%, acidic, 16%, and that half-cysteine was present. Sodium dodecyl sulfate-polyacrylamide gel patterns of solubilized cytoplasmic membrane proteins revealed major bands at 195, 74.5, 44, 32, and 30 KDa. Significant amounts of nickel co-isolated with the cytoplasmic membrane, accounting for 0.16% of the membrane dry weight.

A comparative study on the occurrence and activity of extracellular matrix vesicles in young and adult rat maxillary bone

The occurrence of matrix vesicles in the maxillary bone of normal young and adult rats was demonstrated by both ultrastructural and enzymatic studies. Transmission electron microscopy revealed that the vesicles are invested in trilaminar membranes. Occasionally, crystals were found both within the vesicles and in the surrounding matrix. Separated fractions of vesicles, membranes, and cells were studied biochemically. The amounts of vesicular protein and enzymatic activity per gram bone in the adult rats were significantly lower than in the young. This coincides with the higher number of vesicles observed in the TEM specimens of young rats when compared to that in the old ones. The specific activities of all enzymes examined in the three bone fractions obtained from both young and adult rats were similar. This indicates that no significant difference exists in the enzymatic characteristics of matrix vesicles from the maxillary bone of young and adult rats.

Monocyte-mediated serum-independent damage to hyphal and pseudohyphal forms of Candida albicans in vitro.

Human peripheral blood monocytes attached to Candida albicans hyphae in the absence of serum and damaged the hyphae without completely ingesting them. Attachment and damage was not augmented by the addition of serum. Damage to hyphae was quantitated by a previously developed metabolic assay that measured leukocyte-induced reduction in uptake of [(14)C]cytosine by the hyphae. Use of cells from patients with hereditary disorders of leukocyte function, chronic granulomatous disease, and myeloperoxidase deficiency indicated that myeloperoxidase-independent and nonoxidative mechanisms could sometimes damage hyphae where oxidative mechanisms were impaired. Damage to hyphae by normal monocytes was inhibited by concentrations of sodium azide and sodium cyanide that primarily affect myeloperoxidase activity, as well as by halide-free conditions, catalase, and putative antagonists of hypochlorous acid or singlet oxygen. Iodination of hyphae, a myeloperoxidase and hydrogen peroxide-dependent process of monocytes, was similarly inhibited by sodium azide, sodium cyanide, and catalase. Under anaerobic conditions, damage to hyphae was reduced by 64.0-68.4%. In contrast, inhibitors of potential nonoxidative antifungal mechanisms, iron salts to saturate iron chelators, and polyanionic amino acid polymers to neutralize cationic proteins did not block damage to hyphae by monocytes. Preparations rich in lysosomal granules from fractionated normal monocytes also did not damage hyphae. Overall, it appeared that oxidative mechanisms were most important for damage to hyphae by normal monocytes. Electron microscopy confirmed that Candida hyphae were damaged and probably killed by monocytes, but monocytes appeared to sustain significant damage in the process. In the absence of serum, monocyte cell membranes became closely approximated to Candida cell walls. It appeared that some Candida could escape this partial engulfment, as they were seen floating free with vesicular trilaminar membrane remnants covering hyphal surfaces. In general, monocytes appeared to be damaged by interactions with Candida hyphae more than neutrophils had been in previous studies.

Angiotensin I converting enzyme (kininase II) of the brush border of human and swine intestine

Mucosal brush border of human and swine small intestine is rich in angiotensin I converting enzyme or kininase II (ACE). The brush border of the intestinal mucosa was purified by centrifugation over a discontinuous glycerol gradient. Transmission electron micrographs showed that 90 per cent of the isolated vesicles had a trilaminar membrane structure and glycocalyx, characteristic of intestinal brush border. No significant contamination by other subcellular particles was evident. In the final purified preparation, the brush border marker enzymes sucrase, trehalase and alkaline phosphatase were enriched 23-, 18- and 17-fold from human intestine and 27-, 26- and 20-fold from swine tissue. ACE was highly concentrated in the human and swine brush border. The specific activity of ACE in the human and swine brush border fractions was enriched 17- and 7.6-fold over the crude homogenate. Kininase activity was demonstrated by bioassay. Captopril, the orally active specific inhibitor of ACE, inhibited the enzyme; its I 50 was 3 × 10 −9. Antibody to swine kidney ACE cross-reacted with swine intestinal enzyme as shown in rocket immunoelectrophoresis, indicating that the enzymes from kidney and from intestine have common antigenic determinants and that the enzyme is concentrated on the brush border membrane. Because of the abundant presence of ACE in the intestine, interference in the functions of this enzyme may occur with chronic captopril therapy.

Prolymphocytic leukemia. An ultrastructural study.

The ultrastructural features of the leukemic cells from three patients with prolymphocytic leukemia were examined. In all patients, the prolymphocytes were characterized by a single prominent nucleolus and a chromatin structure intermediate between that of a mature lymphocyte and a lymphoblast. The cytoplasm contained a few short segments of rough endoplasmic reticulum and a poorly-developed Golgi apparatus. Polyribosomes and lysosomes were scarce. These ultrastructural features help in distinguishing prolymphocytic leukemia from other malignant lymphoproliferative disorders. A unique feature of the leukemic cells from one patient was the presence of intracytoplasmic inclusions. These were concentrated in the region of the Golgi zone, were surrounded by a smooth, trilaminar membrane, and had a substructure of parallel lines with a periodicity of approximately 60 A. To the authors' knowledge, similar inclusions have not been previously described in prolymphocytic leukemia.

Immunochemistry of herpes simplex virus glycoproteins.

Herpes simplex viruses (HSV) are large DNA viruses belonging to the Herpesvirus family. Like all other herpesviruses, the virions of HSV are made up of a DNA core, an icosahedral capsid consisting of 162 capsomers, an ill-defined structural component designated the tegument, and a trilaminar membrane containing numerous short spikes, designated the envelope (Fig. 1) (Roizman and Furlong 1974).

Electron microscopical study of Neisseria meningitidis releasing various amounts of free endotoxin.

A study has been made of the ultrastructure of four strains of Neisseria meningitidis which liberate varying amounts of free endotoxin in a chemically-defined, protein-free medium. The two strains which did not release detectable or only sparse amounts of free, filtrable endotoxin were rather uniform in cell size. Their cells appeared to be intact and showed a low tendency to aggregate, In addition cells of these strains showed only sporadic loose, trilaminar membranes and blebs, and free membranous structures were sparse in the medium. The endotoxin releasing strains liberated a high yield of free structures from the outer cell wall into the medium. These structures may represent the lipopolysaccharide (LPS).

Rhabdospora thelohani Laguessé, 1895 (Apicomplexa): new host and geographic records with taxonomic considerations.

New fish species and geographic records for Rhabdospora thelohani Laguessé, 1895 (rodlet cells) are presented. Additionally, the ultrastructure of R. thelohani in Alburnoides bipunctatus ohridanus Karaman, Borostomias antarcticus (Lönnberg), Leuciscus cephalus albus Bonaparte and Rutilus rubilio (Bonaparte) is compared with that reported by other authors and with members of Subphylum Apicomplexa. The ultrastructure of R. thelohani was similar in all the fish species examined; however, the organism was not present in all members of any single species and had intertissue density variations. Rhabdospora thelohani is pyriform, averaging in size 7 X 12 micrometer, with a basal nucleus. The surface complex is composed of a layer (0.5 micrometer diameter) formed by microfilaments (9.3 nm) and an outer trilaminar membrane (9.3 nm). The cytoplasm contains structures identical to rhoptries, micronemes and subpellicular microtubules. Mitochondria, Golgi apparatus, and rough endoplasmic reticulum were not observed, althouth free ribosomes were present and arranged in a vesicular pattern. The observations suggest that the organism moves between cell of epithelial layers and is either released into a lumen intact or passively or actively discharges its contents into a lumen. Results from this study indicate that R. thelohani should be considered a member of Apicomplexa unless definitive evidence is presented to the contrary.

Mammalian central nervous system axolemma: histochemical evidence for axonal plasma membrane origin of bovine and rat axolemma-enriched membrane fractions

Purified myelinated axons were isolated from fresh bovine corpus callosum and rat brainstem and osmotically shocked to strip myelin and axolemma from axon filaments. Two axolemma-enriched fractions, myelin, and a pellet of myelin-free axons were harvested by discontinuous sucrose centrifugation of the shocked myelinated axons. By electron microscopy the axolemma-enriched fraction was composed of trilaminar membrane vesicles (diameter 50–1000 nm) and linear membrane fragments; myelin fragments were seldom seen but some mitochondria) membrane contamination was evident. The classical myelin stain Luxol Fast Blue never counter-stained the myelin-free axons, but did stain myelin, myelinated axons, and to a lesser extent, both axolemmal fractions. The staining of the axolemma-enriched fraction was amorphous and seemed incommensurate with the level of myelin contamination. Acetylcholinesterase staining of the axolemma-enriched fraction counterstained with Luxol Fast Blue revealed a number of acetylcholinesterase-positive membrane vesicles by light microscopy. Occasional enzyme-positive vesicles were also evident in the myelin while myelin-free axons showed virtually no Luxol Fast Blue stain but were heavily stained for the enzyme. Higher resolution histochemical staining by electron microscopy confirmed the light microscopy, showing acetylcholinest erase reaction product lining the axolemma of bovine and rat CNS neurons. Myelin, mitochondria, and other diverse membranes showed no reaction product. About 30% of bovine corpus callosal axons showed some acetylcholinesterase staining but only 3% of the total lengths of all the bovine CNS axolemma showed reaction product. Positive acetylcholinesterase staining could also be demonstrated in a small proportion of the bovine axolemma-enriched membranes, with reaction product lining the membrane fragments and captured within their vesicles. The percentage of vesicles that were stained closely agreed with the percentage of axolemma) length stained in the whole white matter. Since acetylcholinesterase staining was highly localized to the axonal plasma membrane, the acetylcholinesterase-positive membrane fragments in the axolemma-enriched membranes could only originate from the axolemma. We conclude on the basis of the operational behavior of these axolemmal vesicles that many of the membranes found in these fractions are derived from the axonal plasma membrane.

Physicochemical and in situ observations on the adhesion of gliding bacteria to surfaces

The ability of Flexibacter BH3 to adhere to solid surfaces and to overcome the horizontal drag involved in gliding across the surfaces was considered in terms of the Stefan adhesion principle. The extracellular slime produced by Flexibacter BH3 was suitable as a Stefan adhesive because it exhibited viscous properties characteristic of a linear colloid, increasing the adhesiveness of the bacterium but allowing translational motion across the surface. The water-soluble slime was a glycoprotein, containing glucose, fucose, galactose and some uronic acid. Vesicles and tubules on the outer surface of Flexibacter BH3 possessed trilaminar membranes, contained 2-keto-3-deoxyoctonate (KDO), and showed identity with phenol-extracted lipopolysaccharide (LPS) in gel-diffusion tests. Sections of Flexibacter BH3 gliding on a gold film overlaying an agar medium reveraled a highly convuluted cell envelope outer membrane, portions of which closely conformed to the microcontours of the gold surface. Possible mechanisms of gliding are discussed in relation to this close association with solid surface features, to the finding that flexibility and spiral motion are not essential for gliding, and to evidence revealing the extrusion of slime in advance of “pathfinder” bacteria.

Deviated formation of intestinal glycocalyx in human stomach cancer cells

The process of glycocalyx formation by the trilaminar membrane was investigated at the subcellular level by use of cultivated cancer cells derived from a human stomach adenocarcinoma. Glycocalyx was apparently synthesized on the characteristic trilaminar membrane of Golgi-derived vesicles which gave rise to cytoplasmic vacuoles which, in turn, fused to form an intracytoplasmic cyst. Characteristic microvilli similar to those of intestinal epithelium extended from the membrane lining the intracytoplasmic cyst. These ultrastructural features agree with earlier histochemical findings in suggesting intestinal metaplasia in the origin of the gastric tumor. The morphologic features of the cancer cells clearly indicated that glycoprotein is first synthesized in the Golgi complex and fully formed mucoprotein then emerges as membrane-bound glycocalyx in the vesicles budding from the Golgi stacks. The glycocalyx layer is an integral part of the external leaflet of the characteristic trilaminar membrane. Abundant deposits of glycocalyx in the intracytoplasmic cyst constituted the ultrastructural basis for a distinctive type of signet ring cell that differed from mucous signet ring cells derived from goblet cells.

Localization of intestinal sucrase-isomaltase complex on the microvillous membrane by electron microscopy using nonlabeled antibodies.

Microvillous vesicles isolated from rabbit small intestine showed a trilaminar membrane with a rather smooth surface, which was apparently not affected by papain solubilizing sucrase-isomaltase complex or by trypsin unable to solubilize it. When microvilous vesicles or trysinized ones were incubated with immunoglobulin G against the sucrase-isomaltase complex or monovalent fragments therefrom, an apparently continuous electron-opaque layer approximately 180 A in width appeared around the external surface of vesicles. Such a layer was not formed on papainized vesicles. Microvillous vesicles and trypsinized ones negatively stained with phosphotungate showed a great number of particles protruding approximately 150 A from the membrane surface, but papainized vesicles did not. The particles existed close to one another and appeared to form a particulate layer 150 A in width on the surface. The antibodies, whether they were divalent or monovalent, increased the width of the layer to approximately 200 A and obscured the fine particulate structure of intact and trypsinized vesicles. Papainized vesicles retained their smooth surface upon interaction with antibodies. These results, together with those with the Triton-solubilized sucrase- isomaltase complex (Nishi and Takesue, 1978), J. Ultra-struct. Res., 62:1- 12), indicate not only that sucrase-isomaltase complexes are located close to one another on the membrane, but also that they or at least their protein portions protrude approximately 150 A from the surface of the trilaminar membrane.