Morphological studies on the contents of the pulmonary alveolus. Ultrastructural research on thin sections and replicas

The study concerns the contents of the rat alveolus during the course of development. This contents in the fetus is composed exclusively of lamellar bodies, with tubular myelin figures connected to them; in the neonates is composed also of material layered on the surface of the alveolar cells which is initially filamentous, osmiophilic and ruthenium red-negative, then, later on, homogeneous and ruthenium red-positive.     

A peculiar mode of formation of the surface lining layer in the lungs of Salamandra salamandra

The pneumocytes of the larva of Salamandra salamandra contain numerous lamellar bodies and their precursors: electron-dense bodies at various stages of development. Both lamellar bodies and electron-dense bodies occur inside the fluid-filled lung. The former are spherical or bell-shaped and possess concentrically arranged smooth membranes, 8 nm thick; the latter have paracrystalline cores composed of alternately oriented clear and dark striations (3.6–3.9 nm and 2.6–3.6 nm, respectively). On all sides such cores separate membranes, which assume a concentric orientation. No tubular myelin was observed in any phase of the transformation of lamellar bodies and electron-dense bodies into the surface lining layer. Fixation of the lungs of adult individuals with tannic acid-containing fixative visualized the surface lining layer, but not tubular myelin.     

Lysozyme is an ozone-sensitive component of alveolar type II cell lamellar bodies

Exposure of rats to 3 ppm ozone for up to 8 h results in significant changes in lamellar bodies, the surfactant storing organelles of type II cells. We have previously shown that a 14 kDa lamellar body protein is decreased as early as 4 h after the onset of ozone exposure. We have isolated this ozone-sensitive protein from rat lung lamellar bodies and identified it as lysozyme by immunochemical methods, as well as by its amino acid composition, N-terminal amino acid sequence and bacteriolytic activity. Reduced lysozyme activity in isolated lamellar bodies is detected as early as 4 h after the start of ozone exposure. Following an 8 h ozone exposure, the activity does not return to control levels for at least 48 h. Lamellar body lysozyme is expected to be secreted with surfactant phospholipids, thereby contributing to the antimicrobial defense of the alveolar lining layer. The acute lysozyme deficiency seen in ozone-induced oxidant injury may reduce the resistance of the lung to infection.     

Cellular differentiation in the kidneys of newborn mice studies with the electron microscope

The structure of the kidney of the Swiss albino mouse changes progressively during the first 2 weeks after birth. Cells proliferate to form new nephrons, cells differentiate by acquiring specialized membranous components, and certain cytological features which are present at birth diminish in abundance or disappear. The differentiation of the cells of the cortical tubules has been studied using the light and electron microscopes. The tubules are partially and variably differentiated at birth. During the first 2 weeks after birth the brush border develops in the proximal tubules by the accumulation of numerous microvilli on the apical cell margins. Basal striations develop in proximal and distal tubules as an alignment of mitochondria, the result of what appears to be progressive interlocking of adjacent fluted cells. The mitochondria increase in number and size, accumulate homogeneous matrix, and acquire small, very dense granules. The collecting ducts develop tight pleating of the basal cell membranes, and dark cells containing numerous small cytoplasmic vesicles and microvilli appear. At birth there are dense irregular cytoplasmic inclusions presumed to be lipide in renal cells, the cytoplasmic granules of Palade are abundant, and there are large round bodies in the cells of the proximal tubules. The lipide inclusions disappear a few days after birth, and the cytoplasmic granules of Palade diminish in abundance as the cells differentiate. The large round bodies in the proximal tubules consist of an amorphous material and contain concentrically lamellar structures and mitochondria. They resemble the cytoplasmic droplets produced in the proximal tubules of adult rats and mice by the administration of proteins. The large round bodies disappear from the proximal tubules of infant mice during the first week after birth, but the concentric lamellar structures may be found in adult mice.     

The subcellular location, maturation and response to increased plasma glucagon of Ruthenium Red-insensitive calcium-ion transport in rat liver

1. The subcellular distribution and maturation of Ruthenium Red-insensitive Ca(2+) transport activity were determined in livers of rats ranging in age from 3 days pre-term to 10 weeks of adult life and compared with those of glucose 6-phosphatase, 5'-nucleotidase and Ruthenium Red-sensitive Ca(2+) transport. Initial rates of Ruthenium Red-insensitive Ca(2+) transport were highest in those fractions enriched in glucose 6-phosphatase, i.e. the microsomal fraction; this fraction was devoid of Ruthenium Red-sensitive Ca(2+) transport activity. Although the heaviest fraction (nuclear) contained significant amounts of 5'-nucleotidase activity it was devoid of Ruthenium Red-insensitive Ca(2+) transport activity. 2. Foetal rat liver contain minimal amounts of Ruthenium Red-insensitive Ca(2+) transport activity, glucose 6-phosphatase and 5'-nucleotidase activities. These begin to be expressed concomitantly soon after birth; Ruthenium Red-insensitive Ca(2+) transport is maximal by 3 to 4 days and remains so for up to at least 10 weeks of adult life. Glucose 6-phosphatase also reaches a peak at 3-4 days, but then rapidly decreases to approach adult values. Maximal activity of 5'-nucleotidase in the microsomal and nuclear fractions is seen about 4-6 days after birth; this enzyme activity remains increased for up to about 10 days and then falls, but not as rapidly as glucose 6-phosphatase. It is tentatively suggested that the bulk of the Ruthenium Red-insensitive Ca(2+) transport is attributable to the system derived from the endoplasmic reticulum. 3. Administration of glucagon to adult rats enhances by 2-3-fold the initial rate of Ruthenium Red-insensitive Ca(2+) transport in the intermediate but not the microsomal fraction. The hormone-induced effect is fully suppressed by co-administration of puromycin, is dose-dependent with half-maximal response at approx. 1mug of glucagon/100g body wt. and time-dependent exhibiting a half-maximal response about 1h after administration of the hormone. 4. Ruthenium Red-insensitive Ca(2+) transport in the post-mitochondrial fraction of foetal liver also responds to the administration in situ of glucagon. The response, which also is prevented by co-administration of puromycin, is maximal in those foetuses nearing term. The suggestion is made that these effects of the hormone on Ruthenium Red-insensitive Ca(2+) transport are an integral part of the physiological network in the liver cell.     

An electron microscopic study of the acid mucosubstance lining the alveoli of hamster lung

Synopsis Hamster lung has been investigated by electron microscopy using colloidal iron oxide and Ruthenium Red stains. A continuous layer of acid mucosubstance has been demonstrated on the surface of the alveolar epithelial cells. The lining layer associated with the membranous pneumonocytes has been found to be consistently thinner than that covering the granular pneumonocytes and alveolar macrophages. Strong binding of stain has also been observed in osmiophilic membranous material which is thought to represent fragments of pulmonary surfactant. The susceptibility of the alveolar lining material to digestion by neuramidase suggests that it consists mainly of a sialomucin.     

The ontogeny and distribution of surfactant protein B in human fetuses and newborns.

The distribution of immunoreactive surfactant-associated protein B (IR-SP-B) was studied immunohistochemically in 120 subjects from 10 weeks of gestation to 7 postnatal months with a polyclonal antibody against human SP-B. Electron microscopy (EM) was done in 72 subjects to document the presence of Type II cells containing lamellar bodies. Fetuses of less than 18 weeks' gestation showed no immunostaining. Beginning at 18 weeks, non-mucous cells of tracheal glands immunostained in a few instances. Fetuses of 19 through 23 weeks showed progressive immunostaining of cells lining terminal airways. Infants 26-40 weeks who died with or without pulmonary pathology showed immunostaining of Type II cells and bronchioloalveolar (BA) portal cells of the respiratory bronchioles. In infants with hyaline membrane disease (HMD) who died less than 12 days after birth, occasional tracheal gland cells, BA portal cells, and mature and relining Type II cells immunostained. In bronchopulmonary dysplasia (BPD), BA portal cells, relining Type II cells, macrophages, and luminal material immunostained. Occasional tracheal and bronchial gland cells and Clara cells immunostained. The appearance of IR-SP-B at mid-gestation correlated with differentiation of Type II cells. There was good correlation of immunostaining with the presence of lamellar bodies on EM. Accelerated maturation of the lung was often associated with premature rupture of membranes (PROM).     

Annulate lamellae,lamellar bodies and subsurface cisternae in neurons of the avian hyperstriatum accessorium

Summary Structures identified as annulate lamellae, lamellar bodies and subsurface cisternae were found in neurons of the hyperstriatum accessorium of the avian forebrain. Annulate lamellar arrays with up to six lamellae were present in the larger somata. The lamellae were made up of fused smooth-surfaced cisternae forming pores or annuli and were surrounded by a dense filamentous to granular material. Stacks of nonfenestrated, parallel, regularly spaced cisternae, designated as lamellar bodies, also appeared in the cytoplasm. When flattened they were reminiscent of the electron dense subsurface cisternae. Continuity could be demonstrated between peripherally located subsurface cisternae and lamellar bodies. The dense filamentous to finely granular substance was also located between these structures. Annulate lamellae, lamellar bodies and subsurface cisternae were always observed in conjunction with the rough endoplasmic reticulum. The functional significance of these structural associations is considered.     

Methods of heavy metal electron microscopic histochemistry applied to frog lung surfactant.

Four heavy metal staining methods have been applied to frog lung surfactant. Among them, the iodoplatinate method is the only one that almost exclusively visualizes the phospholipid moiety being produced in the lamellated bodies of the pulmonary epithelial cells and forming the backbone of organized structures within the extracellular lining layer. The other three techniques-ruthenium red-osmium tetroxide, osmium tetroxide-ferrocyanide, acidic phosphotungstic acid in chromatic (Rambourg technique)--more or less give electron contrast to glycoproteins and to a lesser extent to the hydrophilic parts of phospholipids. They all show the extracellular lining layer to be a two component system: the content of the lamellar bodies form--when released--membranous configurations, similar to those observed in mammalian lungs; they unfold in an amorphous hypophase, which is apparently secreted by goblet cells of the pulmonary epithelium.     

CELLULAR DIFFERENTIATION IN THE KIDNEYS OF NEWBORN MICE STUDIED WITH THE ELECTRON MICROSCOPE

The structure of the kidney of the Swiss albino mouse changes progressively during the first 2 weeks after birth. Cells proliferate to form new nephrons, cells differentiate by acquiring specialized membranous components, and certain cytological features which are present at birth diminish in abundance or disappear. The differentiation of the cells of the cortical tubules has been studied using the light and electron microscopes. The tubules are partially and variably differentiated at birth. During the first 2 weeks after birth the brush border develops in the proximal tubules by the accumulation of numerous microvilli on the apical cell margins. Basal striations develop in proximal and distal tubules as an alignment of mitochondria, the result of what appears to be progressive interlocking of adjacent fluted cells. The mitochondria increase in number and size, accumulate homogeneous matrix, and acquire small, very dense granules. The collecting ducts develop tight pleating of the basal cell membranes, and dark cells containing numerous small cytoplasmic vesicles and microvilli appear. At birth there are dense irregular cytoplasmic inclusions presumed to be lipide in renal cells, the cytoplasmic granules of Palade are abundant, and there are large round bodies in the cells of the proximal tubules. The lipide inclusions disappear a few days after birth, and the cytoplasmic granules of Palade diminish in abundance as the cells differentiate. The large round bodies in the proximal tubules consist of an amorphous material and contain concentrically lamellar structures and mitochondria. They resemble the cytoplasmic droplets produced in the proximal tubules of adult rats and mice by the administration of proteins. The large round bodies disappear from the proximal tubules of infant mice during the first week after birth, but the concentric lamellar structures may be found in adult mice.     

Methods of heavy metal electron microscopic histochemistry applied to frog lung surfactant

Summary Four heavy metal staining methods have been applied to frog lung surfactant. Among them, the iodoplatinate method is the only one that almost exclusively visualizes the phospholipid moiety being produced in the lamellated bodies of the pulmonary epithelial cells and forming the backbone of organized structures within the extracellular lining layer. The other three techniques — ruthenium red-osmium tetroxide, osmium tetroxideferrocyanide, acidic phosphotungstic acid in chromate (Rambourg technique) — more or less give electron contrast to glycoproteins and to a lesser extent to the hydrophilic parts of phospholipids. They all show the extracellular lining layer to be a two component system: the content of the lamellar bodies from — when released — membranous configurations, similar to those observed in mammalian lungs; they unfold in an amorphous hypophase, which is apparently secreted by goblet cells of the pulmonary epithelium.     

Glucagon stimulation of ruthenium red-insensitive calcium ion transport in developing rat liver.

The maturation of glucagon-stimulated Ruthenium Red-insensitive Ca2+-transport activity was determined in livers of rats ranging in age from 5 days preterm to 10 weeks of adult life. Previous indications are that this activity is confined to vesicles derived mainly from the endoplasmic reticulum. Perinatal-rat liver contains near-adult values of Ruthenium Red-insensitive Ca2+-transport activity, and exhibits large transient increases in the rate of this activity at two stages of development, immediately after birth, and at 2-5 days after birth. The administration of glucagon to foetal rats, at developmental stages after 19.5 days of gestation (2.5 days before birth), results in a large stable increase (greater than 100%) of Ca2+-transport activity in a subsequently isolated 'heavy' microsomal fraction. That this fraction was enriched in vesicles derived from the rough endoplasmic reticulum was indicated by both an electron-microscopic examination and a marker-enzyme analysis of the subcellular fractions. The administration of glucagon into newborn animals only hours old does not enhance further the initial rate of Ca2+-transport activity, and from day 1 to 10 weeks after birth the administration of the hormone results in the moderate enhancement of Ca2+ transport. Experiments with cyclic AMP and inhibitors of phosphodiesterase activity suggest that cyclic AMP plays a key role in the enhancement by glucagon of Ruthenium Red-insensitive Ca2+ transport, and arguments are presented that this transport system has an important metabolic role in the redistribution of intracellular Ca2+ in liver tissue.     

Ultrastructural observations on life cycle stages of Pneumocystis carinii

An ultrastructural study of life cycle stages of Pneumocystis carinii in infected rat lungs in situ was undertaken utilizing 8 different modes of fixation. Three of the fixatives employed gave good fixation of cysts and intracystic bodies, but for the trophic forms fixation was only fair. Both the trophic forms and intracystic bodies have nuclear pores. The mitochondria of the organism have cristae that appear lamellar. One of the fixation modes revealed a thin, electron-dense layer on the outer surface of the cell wall, a "fuzzy coat" that had not been described previously. This material appears to mediate tight adhesion of trophic forms with other trophic forms, cysts, and with pneumocytes of the lung alveolus.     

Ultrastructural Observations on Life Cycle Stages of Pneumocystis carinii

An ultrastructural study of life cycle stages of Pneumocystis carinii in infected rat lungs in situ was undertaken utilizing 8 different modes of fixation. Three of the fixatives employed gave good fixation of cysts and intracystic bodies, but for the trophic forms fixation was only fair. Both the trophic forms and intracystic bodies have nuclear pores. The mitochondria of the organism have cristac that appear lamellar. One of the fixation modes revealed a thin, electron-dense layer on the outer surface of the cell wall, a "fuzzy coat" that had not been described previously. This material appears to mediate tight adhesion of trophic forms with other trophic forms, cysts, and with pneumocytcs of the lung alveolus.     

Electron microscopy studies of the limiting layers of the rickettsia Coxiella burneti.

Surface layers of Coxiella burneti studied at a high resoulution reveal a plasma membrane and an outer surface membrane 6 to 7 nm thick, and a thin, moderately electron-dense intermediate layer associated with the inner surface of the outer membrane of many cells. This layer appears to be unaffected by lysozyme treatment. Ruthenium red staining was used to delineate a layer of filamentous material external to the outer membrane; this fuzzy layer has a mean thickness of 20 nm and is not often seen on the surface of cells prepared by conventional means. Both antigenic phase I and II cells show a ruthenium red-binding surface layer. It is suggested that this fuzzy layer may be, among other possibilities, a highly branched mucopolysaccharide.     

The effect of carnosine on the healing of a lung wound

The influence of carnosine (beta-alanine, alpha-histidine) on the process of lung wound reparation was studied in 90 guinea-pigs. Its efficacy was evaluated microscopically, histologically and by means of electronic microscope. It was established that carnosine, as compared to controls nearly twice accelerates reparative processes in the injured lung by activation of fibroblast proliferation, connective tissue generation and intracellular regeneration. In type II epithelial cells more intensive formation of osmiophilic bodies and lamellar bodies contained in them is observed. The alveolus formation in the wound lips by the 7-8th day after lung injury is going on under the carnosine influence, which is due, probably, to massive excretion of osmiophilic contents from type II epithelial cells and surfactant production. This may be connected with rapid decrease of atelectasis and rapid restoration of lung airness within the wound.     

A 3H-thymidine autoradiographic study of the development of the cerebellum of the White Leghorn (Gallus domesticus): "evidence for longitudinal neuroblast generation patterns"

Dynamic aspects of the development of the cerebellum of the white Leghorn (Gallus domesticus) were examined using 3H-thymidine autoradiography. A single dose of 25 microCi 3H-thymidine was given to embryos varying in age from 0-18 days. The embryos were all studied before hatching, either after short or after long survival times. It appeared that the superficial part of the outer mantle layer (OML-s, or called external granular layer, egl) is histologically present at day 6, but starts its activity as a secondary matrix at day 7, probably due to activation by a secondary influx of cells from the ventricular neuro-epithelium. It was also demonstrated that Purkinje cells and cells of the central cerebellar nuclei are produced by the ventricular neuro-epithelium mainly at days 3, 4 and 5. No birth of these cells was observed beyond day 5. In addition, a relationship was found between the birth patterns of Purkinje cells and cells of the central cerebellar nuclei and the longitudinal cytoarchitectonic patterns as observed in normal Nissl stained material. It was concluded that these birth patterns are an early contribution to the establishment of the longitudinal organization of the cerebellum.     

The phospholipids of lamellar body material from fetal rabbit lung tissue in culture. Unusual phosphatidylcholine fatty acid profile

Tissue pieces of rabbit fetal lung, 23 days gestation, were cultured for 7 days in serum-free medium to obtain lamellar body material for phospholipid analysis. Cultures were maintained in culture medium without serum and (1) with no added hormones (control cultures), (2) with thyroxine (1 x 10(-7) M), (3) with cortisol (1 x 10(-7) M) and (4) with thyroxine plus cortisol (1 x 10(-7) M each). The hormonal response was evaluated by measuring the quantity of lamellar body material isolated from the tissue pieces after the 7-day culture period. Compared to control cultures, more lamellar body material was recovered from cultures treated with cortisol (180% of control) and with thyroxine plus cortisol (250% of control). Cultures treated with thyroxine alone yielded the same amount of lamellar body material as the controls. Hormone treatment produced only minor changes in the glycerophospholipid profile of the lamellar body material. A small but significant increase in the percentage of phosphatidylglycerol and a small but significant decrease in phosphatidylinositol were found in lamellar body material from cultures treated with thyroxine and thyroxine plus cortisol. The disaturated phosphatidylcholine content of the lamellar body material from culture was 28% of the total lamellar body phospholipid and was not affected by hormone treatment. This disaturated phosphatidylcholine content was low compared to the disaturated phosphatidylcholine of lamellar body material from adult lung (46%). The low proportion of disaturated phosphatidylcholine was due to the unusual presence of palmitoleic acid (16:1(cis-9)), which was more than one-fourth of the total fatty acid of the lamellar body phosphatidylcholine. It is possible that an abnormal delta 9 fatty acid desaturation activity was expressed in the lung tissue in vitro, which resulted in the high incorporation of the 16:1 fatty acid into lamellar body phosphatidylcholine.     

Cytochemical study of the lamellar bodies in the swimbladder of the toadfish Opsanus tau L.

Summary The columnar epithelial cells of the gas gland in the swimbladder of the toadfish, Opsanus tau L., contain lamellar bodies that resemble the lamellar bodies found in epithelial cells of vertebrate lungs. Cytochemical assays indicate that swimbladder lamellar bodies are soluble in chloroform-methanol solution, react with tricomplex flocculation solution (indicating a phospholipid component), exhibit a positive reaction for cholesterol when exposed to digitonin, and contain acid phosphatase.The anterior chamber of the toadfish swimbladder is lined by an extracellular layer. Digitonin-cholesterol crystals are found in this layer when the swimbladder is treated with digitonin. A ruthenium red positive layer is also present in the anterior chamber of the toadfish swimbladder.The structure and cytochemistry of swimbladder lamellar bodies are compared with those of vertebrate lung lamellar bodies. Similarities between the extracellular layer in the swimbladder and the extracellular layer in lungs are also noted.Supported in part by a grant No 1 R23 HL 19593-01 from the National Institutes of Health     

ULTRASTRUCTURE OF CARPOSPOROGENESIS IN THE PARASITIC RED ALGA FAUCHEOCOLAX ATTENUATA SETCH. (RHODYMENIALES,RHODYMENIACEAE)

Carpospore differentiation in Faucheocolax attenuata Setch. can be separated into three developmental stages. Immediately after cleaving from the multinucleate gonimoblast cell, young carpospores are embedded within confluent mucilage produced by gonimoblast cells. These carpospores contain a large nucleus, few starch grains, concentric lamellae, as well as proplastids with a peripheral thylakoid and occasionally some internal (photosynthetic) thylakoids. Proplastids also contain concentric lamellar bodies. Mucilage with a reticulate fibrous substructure is formed within cytoplasmic concentric membranes, thus giving rise to mucilage sacs. Subsequently, these mucilage sacs release their contents, forming an initial reticulate deposition of carpospore wall material. Dictyosome vesicles with large, single dark-staining granules also contribute to wall formation and may create a separating layer between the mucilage and carpospore wall. During the latter stages of young carpospores, starch is polymerized in the perinuclear cytoplasmic area and is in close contact with endoplasmic reticulum. Intermediate-aged carpospores continue their starch polymerization. Dictyosomes deposit more wall material, in addition to forming fibrous vacuoles. Proplastids form thylakoids from concentric lamellar bodies. Mature carpospores are surrounded by a two-layered carpospore wall. Cytoplasmic constituents include large floridean starch granules, peripheral fibrous vacuoles, mature chloroplasts and curved dictyosomes that produce cored vesicles which in turn are transformed into adhesive vesicles. Pit connections remain intact between carpospores but begin to degenerate. This degeneration appears to be mediated by microtubules.