Multilamellar Body Research Articles

Sigmar1 ablation leads to lung pathological changes associated with pulmonary fibrosis, inflammation, and altered surfactant proteins levels.

Sigma1 receptor protein (Sigmar1) is a small, multifunctional molecular chaperone protein ubiquitously expressed in almost all body tissues. This protein has previously shown its cardioprotective roles in rodent models of cardiac hypertrophy, heart failure, and ischemia-reperfusion injury. Extensive literature also suggested its protective functions in several central nervous system disorders. Sigmar1's molecular functions in the pulmonary system remained unknown. Therefore, we aimed to determine the expression of Sigmar1 in the lungs. We also examined whether Sigmar1 ablation results in histological, ultrastructural, and biochemical changes associated with lung pathology over aging in mice. In the current study, we first confirmed the presence of Sigmar1 protein in human and mouse lungs using immunohistochemistry and immunostaining. We used the Sigmar1 global knockout mouse (Sigmar1-/-) to determine the pathophysiological role of Sigmar1 in lungs over aging. The histological staining of lung sections showed altered alveolar structures, higher immune cells infiltration, and upregulation of inflammatory markers (such as pNFκB) in Sigmar1-/- mice compared to wildtype (Wt) littermate control mice (Wt). This indicates higher pulmonary inflammation resulting from Sigmar1 deficiency in mice, which was associated with increased pulmonary fibrosis. The protein levels of some fibrotic markers, fibronectin, and pSMAD2 Ser 245/250/255 and Ser 465/467, were also elevated in mice lungs in the absence of Sigmar1 compared to Wt. The ultrastructural analysis of lungs in Wt mice showed numerous multilamellar bodies of different sizes with densely packed lipid lamellae and mitochondria with a dark matrix and dense cristae. In contrast, the Sigmar1-/- mice lung tissues showed altered multilamellar body structures in alveolar epithelial type-II pneumocytes with partial loss of lipid lamellae structures in the lamellar bodies. This was further associated with higher protein levels of all four surfactant proteins, SFTP-A, SFTP-B, SFTP-C, and SFTP-D, in the Sigmar1-/- mice lungs. This is the first study showing Sigmar1's expression pattern in human and mouse lungs and its association with lung pathophysiology. Our findings suggest that Sigmar1 deficiency leads to increased pulmonary inflammation, higher pulmonary fibrosis, alterations of the multilamellar body stuructures, and elevated levels of lung surfactant proteins.

Pathogenic variants of Valosin-containing protein induce lysosomal damage and transcriptional activation of autophagy regulators in neuronal cells.

Mutations in the valosin-containing protein (VCP) gene cause various lethal proteinopathies that mainly include inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). Different pathological mechanisms have been proposed. Here, we define the impact of VCP mutants on lysosomes and how cellular homeostasis is restored by inducing autophagy in the presence of lysosomal damage. By electron microscopy, we studied lysosomal morphology in VCP animal and motoneuronal models. With the use of western blotting, real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence and filter trap assay, we evaluated the effect of selected VCP mutants in neuronal cells on lysosome size and activity, lysosomal membrane permeabilization and their impact on autophagy. We found that VCP mutants induce the formation of aberrant multilamellar organelles in VCP animal and cell models similar to those found in patients with VCP mutations or with lysosomal storage disorders. In neuronal cells, we found altered lysosomal activity characterised by membrane permeabilization with galectin-3 redistribution and activation of PPP3CB. This selectively activated the autophagy/lysosomal transcriptional regulator TFE3, but not TFEB, and enhanced both SQSTM1/p62 and lipidated MAP1LC3B levels inducing autophagy. Moreover, we found that wild type VCP, but not the mutants, counteracted lysosomal damage induced either by trehalose or by a mutant form of SOD1 (G93A), also blocking the formation of its insoluble intracellular aggregates. Thus, chronic activation of autophagy might fuel the formation of multilamellar bodies. Together, our findings provide insights into the pathogenesis of VCP-related diseases, by proposing a novel mechanism of multilamellar body formation induced by VCP mutants that involves lysosomal damage and induction of lysophagy.

A New Homotetramer Hemoglobin in the Pulmonary Surfactant of Plateau Zokors (Myospalax Baileyi).

The plateau zokor (Myospalax baileyi) is a native species to the Qinghai-Tibetan Plateau, inhabiting hypoxia and hypercapnia sealed subterranean burrows that pose several unique physiological challenges. In this study, we observed a novel heme-containing protein in the pulmonary surfactant (PS) of plateau zokor, identified the encoding gene of the protein, predicted its origination and structure, verified its expression in alveolar epithelial cells, and determined the protein’s affinity to oxygen and its effect on the oxygen-dissolving capability in the PS of plateau zokors. The protein is an unusual homotetramer hemoglobin consisting of four γ-like subunits, and the subunit is encoded by a paralog gene of γ, that is γ-like. The divergence time of γ-like from γ is estimated by the molecular clock to be about 2.45 Mya. The generation of γ-like in plateau zokors might well relate to long-time stress of the high land hypoxia. Unlike γ, the γ-like has a hypoxia response element (HRE) and a lung tissue-specific enhancer in its upstream region, and it is expressed specifically in lung tissues and up-regulated by hypoxia. The protein is named as γ4-like which is expressed specifically in Alveolar epithelial type II (ATII) cells and secreted into the alveolar cavities through the osmiophilic multilamellar body (LBs). The γ4-like has a higher affinity to oxygen, and that increases significantly oxygen-dissolving capability in the PS of plateau zokors by its oxygenation function, which might be beneficial for the plateau zokors to obtain oxygen from the severe hypoxia environments by facilitating oxygen diffusion from alveoli to blood.

Excessive dietary lipid intake provokes an acquired form of lysosomal lipid storage disease in the kidney.

Obesity and dyslipidaemia are features of the metabolic syndrome and risk factors for chronic kidney disease. The cellular mechanisms connecting metabolic syndrome with chronic kidney disease onset and progression remain largely unclear. We show that proximal tubular epithelium is a target site for lipid deposition upon overnutrition with a cholesterol-rich Western-type diet. Affected proximal tubule epithelial cells displayed giant vacuoles of lysosomal or autophagosomal origin, harbouring oxidised lipoproteins and concentric membrane layer structures (multilamellar bodies), reminiscent of lysosomal storage diseases. Additionally, lipidomic analysis revealed renal deposition of cholesterol and phospholipids, including lysosomal phospholipids. Proteomic profiles of renal multilamellar bodies were distinct from those of epidermis or lung multilamellar bodies and of cytoplasmic lipid droplets. Tubular multilamellar bodies were observed in kidney biopsies of obese hypercholesterolaemic patients, and the concentration of the phospholipidosis marker di-docosahexaenoyl (22:6)-bis(monoacylglycerol) phosphate was doubled in urine from individuals with metabolic syndrome and chronic kidney disease. The enrichment of proximal tubule epithelial cells with phospholipids and multilamellar bodies was accompanied by enhanced inflammation, fibrosis, tubular damage markers, and higher urinary electrolyte content. Concomitantly to the intralysosomal lipid storage, a renal transcriptional response was initiated to enhance lysosomal degradation and lipid synthesis. In cultured proximal tubule epithelial cells, inhibition of cholesterol efflux transport or oxysterol treatment induced effects very similar to the in vivo situation, such as multilamellar body and phospholipid amassing, and induction of damage, inflammatory, fibrotic, and lipogenic molecules. The onset of phospholipidosis in proximal tubule epithelial cells is a novel pathological trait in metabolic syndrome-related chronic kidney disease, and emphasises the importance of healthy lysosomes and nutrition for kidney well-being. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

N370S-GBA1 mutation causes lysosomal cholesterol accumulation in Parkinson's disease.

Heterozygous mutations in the GBA1 gene, which encodes the lysosomal enzyme β-glucocerebrosidase-1, increase the risk of developing Parkinson's disease, although the underlying mechanisms remain unclear. The aim of this study was to explore the impact of the N370S-GBA1 mutation on cellular homeostasis and vulnerability in a patient-specific cellular model of PD. We isolated fibroblasts from 4 PD patients carrying the N370S/wild type GBA1 mutation and 6 controls to study the autophagy-lysosome pathway, endoplasmic reticulum stress, and Golgi apparatus structure by Western blot, immunofluorescence, LysoTracker and Filipin stainings, mRNA analysis, and electron microscopy. We evaluated cell vulnerability by apoptosis, reactive oxygen species and mitochondrial membrane potential with flow cytometry. The N370S mutation produced a significant reduction in β-glucocerebrosidase-1 protein and enzyme activity and β-glucocerebrosidase-1 retention within the endoplasmic reticulum, which interrupted its traffic to the lysosome. This led to endoplasmic reticulum stress activation and triggered unfolded protein response and Golgi apparatus fragmentation. Furthermore, these alterations resulted in autophagosome and p62/SQSTM1 accumulation. This impaired autophagy was a result of dysfunctional lysosomes, indicated by multilamellar body accumulation probably caused by increased cholesterol, enlarged lysosomal mass, and reduced enzyme activity. This phenotype impaired the removal of damaged mitochondria and reactive oxygen species production and enhanced cell death. Our results support a connection between the loss of β-glucocerebrosidase-1 function, cholesterol accumulation, and the disruption of cellular homeostasis in GBA1-PD. Our work reveals new insights into the cellular pathways underlying PD pathogenesis, providing evidence that GBA1-PD shares common features with lipid-storage diseases. © 2017 International Parkinson and Movement Disorder Society.

Long Term Culture of the A549 Cancer Cell Line Promotes Multilamellar Body Formation and Differentiation towards an Alveolar Type II Pneumocyte Phenotype

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.

Screening type II alveolar epithelial cell apoptosis related microRNA

To screen type II alveolar epithelial cell (AECII) apoptosis related microRNA (miRNA) with gene chip technology, provide a new strategy for the prevention and treatment of hyperoxia-induced acute lung injury(HALI). AECII of male Sprague-Dawley (SD) rats was primarily cultured for 36 hours, then exposed to 0.5 mmol/L H2O2 to establish apoptosis model. Transmission electron microscope (TEM) was used to identify AECII and to observe apoptosis cell morphology. Before and after H2O2 injury for 2.5, 6, 12 and 24 hours, fluorescence-activated cell sorting (FACS) was employed to detect apoptosis rate. Additionally, gene chip technology and real time polymerase chain reaction (RT-PCR) were used to screen and verify apoptosis related miRNA respectively. Microvilli and osmiophilic multilamellar body were found under TEM, which were the characteristic structure of AECII. This proved that the cells cultured were AECII. After H2O2 injury for 24 hours, cytoplasmic retraction, chromatin condensation and margination, microvilli and osmiophilic multilamellar body disappearance could be found under TEM. Compared with the blank control group, the apoptosis rate of AECII was significantly increased after exposed to 0.5 mmol/L H2O2, and gradually increased with time [the early apoptosis rate before and after H2O2 injury for 2.5, 6, 12 and 24 hours were (9.43±1.02)%, (18.38±2.91)%, (28.57±1.18)%, (35.83±2.66)% and (57.68±2.22)%, respectively, all P<0.05]. Compared with before H2O2 injury, cells at 24 hours accompanied a significantly changed miRNA expression profiling, in which apoptosis related miRNA had been screened, they were rno-miR-449a-5p, rno-miR-34b/c-5p, rno-miR-200a/c-3p, rno-miR-146a-5p, rno-miR-141-3p, rno-miR-21-5p, rno-miR-375-3p, rno-miR-29b-3p, rno-miR-214-5p, rno-miR-210-5p and rno-miR-214-3p,and the results of RT-PCR were consistent with the gene chip results. Screened out AECII apoptosis related miRNA, and miR-34 family may play a key role in AECII apoptosis regulation. Rno-miR-21-5p may be an important anti-apoptotic gene in AECII apoptosis regulation.

Rescue of murine silica-induced lung injury and fibrosis by human embryonic stem cells

Alveolar type II pneumocytes (ATII cells) are considered putative alveolar stem cells. Since no treatment is available to repair damaged epithelium and prevent lung fibrosis, novel approaches to induce regeneration of injured alveolar epithelium are desired. The objective of this study was to assess both the capacity of human embryonic stem cells (HUES-3) to differentiate in vitro into ATII cells and the ability of committed HUES-3 cells (HUES-3-ATII cells) to recover in vivo a pulmonary fibrosis model obtained by silica-induced damage. In vitro differentiated HUES-3-ATII cells displayed an alveolar phenotype characterised by multi-lamellar body and tight junction formation, by the expression of specific markers such as surfactant protein (SP)-B, SP-C and zonula occludens (ZO)-1 and the activity of cystic fibrosis transmembrane conductance regulator-mediated chloride ion transport. After transplantation of HUES-3-ATII cells into silica-damaged mice, histological and biomolecular analyses revealed a significant reduction of inflammation and fibrosis markers along with lung function improvement, weight recovery and increased survival. The persistence of human SP-C, human nuclear antigen and human DNA in the engrafted lungs indicates that differentiated cells remained engrafted up to 10 weeks. In conclusion, cell therapy using HUES-3 cells may be considered a promising approach to lung injury repair.

Clinical Analysis of the Prognostic Value of Ultrastructural Abnormalities of Plasma Cells in Patients with Multiple Myeloma

The prognostic value of ultrastructural abnormalities of plasma cells was analyzed in 61 patients with multiple myeloma treated in our hospitals from1985 to1996. The survival curves demonstrated significant differences by stratification of ultrastructural abnormalities, such as irregular contour of the nuclear membrane, nuclear body, budding, disarrangement of organelles, fibrillar formation, intramitochondrial granules, multilamellar body, single-sac loop-like structure, and vacuole. However, categorizing by nuclear inclusion body, cytoplasmic inclusion body and dense body showed no significant difference in survival curves. Abnormalities, as well as maturity, may be useful in predicting the prognosis of myeloma patients.

Relationship between ultrastructure of myeloma cells and clinical features in elderly patients with multiple myeloma

The relationship between the ultrastructure of plasma cells and clinical features was analyzed in 54 patients with multiple myeloma: 20 elderly patients (9,8 and 3 cases of IgG, IgA and BJP types, respectively; 5,3 and 12 cases in clinical stage I, II and III, respectively) and 34 young patients (16, 10, 1 and 7 cases of IgG, IgA, IgD and BJP types, respectively; 5, 10 and 19 cases in clinical stage I, II and III, respectively). Five-year survival rates of the elderly and young groups were 41.5 and 60.5%, respectively, though the difference of both survival curves was not significant. Response rates of initial therapy in the elderly and young groups were 55 and 56%, respectively (not significant). The number of patients presenting disarrangement of organelles was significantly larger in the elderly group, but those presenting other abnormal structures were equal between both groups. The ratios of dense body, intramitochondrial granules and multilamellar body in bone marrow plasma cells were significantly higher in nonresponders than in responders of the elderly group. The ratios of disarrangement of organelles, single sac loop-like structure and intramitochondrial granules in bone marrow plasma cells were significantly higher in nonresponders than in responders of the young group. It is suggested that the ultrastructure of plasma cells in multiple myeloma differs between elderly and young patients and is useful in predicting the respective clinical features, although drug responses and survival curves do not show a significant difference.

Two Cases of Juvenile Type Generalized Ceroid‐Lipofuscinosis: An Ultrastructural Study of Storage Material

The clinical course, distribution and morphology of the characteristic inclusions in two cases of generalized ceroid‐lipofuscinosis of juvenile type were investigated. The first case was a Japanese man who died aged 21. He developed visual disturbances at the age of 6 years, and became blind within a year. Epileptic seizures and dementia progressed from the age of 12. The second case was a Japanese woman who also died aged 21 years. She developed dementia, convulsions and visual disturbances at the age of 6 years and progressed gradually. She became almost completely blind at the age of 14 years. In both of the cases, the autopsy revealed diffuse atrophy of the brain. The histology showed numerous lipofuscin‐like granules in the swollen cytoplasm of nerve cells and astrocytes. The storage materials revealed four distinct ultrastructural profiles as follows; 1) multilamellar body (MLB), 2) curvilinear body (CB), 3) crystalloid profile (CP), 4) finger print profile (FPP). The distribution of these storage materials in both central and peripheral nervous system and in various tissue of the body was studied systematically and shown in a table.

Red cell membrane protein distribution during malarial invasion

Immuno-gold labelling electron microscopy of thin sections was used to determine the distribution of red cell membrane and membrane skeleton proteins in the vicinity of internalized malaria parasites. When examined immediately after invasion (young ring-stage parasites), the parasitophorous vacuole membranes of both Plasmodium falciparum and P. knowlesi were found to be characterized by the essentially complete absence of spectrin, ankyrin and the most abundant transmembrane protein, band 3. P. knowlesi merozoites were trapped in the attached but not internalized state by pretreatment with cytochalasin B. In this merozoite-red cell complex antibody labelling showed that band 3 had been eliminated from the region of the host cell membrane in contact with the parasite. Internal vesicles, originating apparently from the site of attachment, were often observed in the red cell. Opposite the attached parasite a cavity was also sometimes seen in the host cell, presumably representing an incipient internal vesicle. The membrane was intact, as judged by the absence of protein (haemoglobin) in the cavity, and, like the membranes surrounding the internal vesicles, was devoid of membrane proteins. A large multilamellar body was sometimes seen in the merozoite close to its point of attachment. The lamellar spacing was about 50 nm. The electron microscope images suggest a diffusion of electron-dense material from the lamellar body into the cavity in the host cell.

Multilamellar body size comparison from lung of embryos and hatchlings of chick

The functional lung of a newborn animal requires surfactant for the purpose of reducing the surface tension at the airtissue interface. Reduced surface tension prevents the lung from collapsing due to high pressures associated with ventilation and lung geometry; and also lowered tension assists in the movement of respiratory gases in and out of the lung tissue. Surfactant first appears late in lung development and correlates with the appearance of a morphological structure in Type II alveolar cells called a multilamellar body. The multilamellar body (LAM) is comprised mainly of phospholipids which serve as the principal ingredient of lung surfactant. The study here reported involves a comparison of multilamellar body size just before and after hatching of white leghorn chick.Eggs of Gallus domesticus, white leghorn breed, were incubated at 99 3/4°F at a relative humidity of 50-56%. Fertile eggs weighing between 50 and 70 grams were divided into two groups: 1) group one eggs were incubated nineteen 24-hour periods before sacrifice and 2) group two eggs were allowed to hatch and sacrificed twenty-two 24-hour periods after the onset of incubation.

Phosphatidate phosphatase activity in the blood fluke, Schistosoma mansoni

Phosphatidate phosphatase activity was found in tissue homogenates and a multilamellar body fraction of Schistosoma mansoni. The identity of the enzyme activity was established on the basis of its pH optimum and inhibition by Mg 2+, Ca 2+ and Tween 20. A seven-fold enrichment of enzyme activity occurred in response to fractionation of the multilamellar bodies. The importance of this enzyme is discussed in terms of its being a marker enzyme and its central role in phospholipid biosynthesis.

Studies on drug-induced lipidosis: subcellular localization of phospholipid and cholesterol in the liver of rats treated with chloroquine or 4,4'-bis (diethylaminoethoxy)alpha, beta-diethyldiphenylethane.

Administration of chloroquine or 4,4'-bis(diethylaminoethoxy)alpha, beta-diethyldiphenylethane (DH) to rats in oral doses of 100 mg/kg for 7 days causes phospholipid and cholesteryl ester accumulation in liver. To further characterize this drug-induced lipidosis, we have isolated and characterized the lipids of subcellular fractions from control rats and rats treated with chloroquine, DH, and Triton WR-1339. The phospholipid content of liver is increased 1.5-fold by chloroquine or DH treatment but is unaffected by Triton WR-1339. Acid phosphatase is increased by treatment with these three agents. Chloroquine and DH cause a shift of acid phosphatase from the light mitochondrial fraction (L) to the heavy mitochondrial fraction (M). Multilamellar bodies, an ultrastructural hallmark of chloroquine and DH-induced lipidosis, were isolated in a highly-purified form from the M fraction of chloroquine- or DH-treated rats. They are highly enriched in acid phosphatase indicating their lysosomal origin. In addition, they contain large amounts of phospholipid, cholesterol, and cholesteryl ester and are the sole site of bis(monoacylglycero)phosphate and the enzyme which catalyzes its synthesis from phosphatidylglycerol. Analysis of the phospholipid content of the respective control and drug-treated liver fractions shows that the entire excess phospholipid content of chloroquine- or DH-treated liver can be accounted for by the drug-induced multilamellar bodies. Triton WR-1339-induced lysosomes, which were isolated for comparison, also contain bis(monoacyglycero)phosphate and bis(monoacyglycero)phosphate synthetase. However, they differ from the drug-induced lysosomes in that their sphingomyelin content is much higher and their total phospholipid and phosphatidylinositol content much lower. The multilamellar bodies are the principal intracellular site of accumulation of chloroquine and DH, respectively. Increased delivery of phospholipid to lysosomes and decreased lysosomal catabolism of phospholipid are the factors which are thought to cause this experimental lipidosis. High levels of phosphatidylinositol in the multilamellar body may be in part responsible for the increased content of bis(monoacyglycero)phosphate since it has been identified as an acyl donor in bis(monoacylglycero)phosphate synthesis.

The ultrastructure of multilamellar bodies and surfactant in the human lung.

Normal tissues from human lungs were dehydrated through Epon 812 resin to retain many of the lipids and carbohydrates in thin section. The three-dimensional structure of the multilamellar body was determined. The paired layer of phospholipid heads (PH) is 36 A thick; the layer of fatty-acid tails (FA) is 31 A, the same as reported previously for non-human primates and rodents. The human multilamellar body is apparently unique: the lamellae of the major focus divide into two or three lamellae; the matrix material of the core is without vesicular bodies and a projection core is present. When compared with those of the rat, human tissues contain a greater number of lamellar foci and fewer lamellae per focus. The presence of a peripheral layer of lamellae, an ever-present external limiting membrane, and the fusion of multilamellar bodies are also characteristic. Tubular myelin surfactant has the same appearance as in other mammals. Multilamellar bodies were observed in direct communication with Golgi vesicles. Their origin from multivesicular bodies and their maturation through secretion and exocytosis were demonstrated. Untransformed multilamellar bodies in the alveolar space demonstrated three periodicities (P): (1) compact regular lamellae, PH = 36 A, FA = 36 A, FA = 31 A, P = 66 A; (2) compact broad lamellae, PH = 72 A, FA = 22 A, P = 94 A; (3) loose lamellae, PH = 36 A, FA = 31 A with a variable interlamellar space.

The periodicity and architecture of lipid retained and extracted lung surfactant and its origin from multilamellar bodies

Lipid and carbohydrate retaining procedures were utilized to retain these components of surfactant in thin section. The origin of tubular myelin surfactant from multilamellar bodies was postulated on morphological evidence to be : an expansion of lamellae in pairs caused by the absorption of a homogeneous, probably carbohydratebased, material of undetermined origin; immediately after the pairs expanded to 438 Å, the lamella (lipid bilayer) that was most peripheral in the multilamellar body underwent a molecular rearrangement to form the two cross lipid bilayers; the four lipid bilayers formed a square tubule 413 Å of side and 0.16–1.8 μ long. The homogeneous material included within each tubule (the surfactant matrix) did not completely fill the intratubular space, but a centrally located, spherical, 57 Å diameter, electron transparent, area was observed. The 413 Å square tubules were separated on all sides by a 25 Å fatty acid tail area. The phospholipid head areas were 46 Å and the surfactant matrix was 132 Å between the electron transparent, center area and the phospholipid heads layer. Extraction with several organic solvents was also studied: slight ethanol extraction—alteration of surfactant matrix and loss of transparent, central area. Moderate ethanol extraction—alteration of surfactant matrix to form artifactual tubules and filaments, disruption of lipid bilayers. Severe extraction—ethanol produced loss of integrity of both matrix and lipid bilayers; vinylcyclohexane dioxide or methacrylate produced complete loss of all lipid bilayers, but surfactant matrix retained tubular configuration ; DMP-30 produced complete loss of lipid bilayers, and although most of the matrix material was retained, all areas appeared fractured and randomly oriented.

The three-dimensional aspect of the mammalian lung surfactant myelin figure

Rodent and primate lung surfactant was studied at the ultrastructural level utilizing procedures that retained most of the carbohydrates and lipids in thin section. The three-dimensional aspect of tubular myelin surfactant was observed to be four, lipid bilayer membranes oriented at right angles so that in cross-section it was square. In longitudinal section it appeared as two parallel lipid bilayers. Inside the tubular myelin was a homogeneous matrix material that completely filled the tubule except for a small, central area. A single multilamellar body, after it expanded and rearranged lamellae to form tubular myelin surfactant, still retained its basic morphology so that it was possible to determine the number and orientation of bodies that comprised a given surfactant area. This enabled quantification of surfactant by serial sectioning. Each transformed multilamellar body was observed to contain from 2 to 13 groups of tubular myelin, oriented at angles within the transformed body. With three-dimensional understanding, many of the areas previously reported to be homogeneous were determined to actually be oblique cross or longitudinal sections through tubular myelin surfactant. Five distinct layers characterized tubular myelin surfactant: (1) Unexpanded layer—up to 63 recently secreted multilamellar bodies. (2) Formation layerp̄aired lamellae expanding and rearranging to form tubules. (3) Mature layer—tubular myelin surfactant. (4) Air-surfactant interface layer—usually a single lipid bilayer which was the outermost layer of tubular myelin of from 1 to 12 transformed multilamellar bodies. (5) Degraded surfactant layer—lipid bilayer spheres were formed at the interface and degraded in the alveolar space.

The three-dimensional aspect of mammalian lung multilamellar bodies

The three-dimensional aspect of rat and monkey lung multilamellar bodies was demonstrated in lipid retained thin sections. The glutaraldehyde and urea lipid retention embedment and an Epon 812 resin polar dehydrant procedure were utilized to retain lamellar lipids for precise morphological study. The unextracted multilamellar bodies were found to conform to a general, though complex, threedimensional structure. A model that demonstrated that structure was derived. Freezeetch and extracted material were shown to support the model. Mature multilamellar bodies were from 1.2–1.6 μ in diameter and were 1.0–1.6 μ high. Each body contained a matrix core that included from 2–25 vesicular bodies and was in contact with the limiting membrane at the matrix plate. Most bodies had from 25–70 lamellae attached for 360 ° to the projection plate. Microtubules were seen in communication with the matrix core. When sectioned in longitudinal section, lamellae projected from the base plate and coursed parallel to the limiting membrane of the top half of the body. Any cross-section produced circular lamellae without apparent attachment. Oblique sections sometimes produced both ‘stacked’ and ‘circular’ lamellae. Four postulates of multilamellar body formation were discussed in light of these findings.

The high resolution ultrastructure of the periodicity and architecture of lipid-retained and extracted lung multilamellar body laminations

High resolution electron microscopy was utilized to study the periodicity and architecture of rat and monkey, lung multilamellar bodies. In lipid retention embedments and polar dehydrants, the lamellar width (LW) was defined as a lipid bilayer. The osmiophilic band that contained two layers of phospholipid heads (PH) of adjacent lipid bilayers in immediate opposition and the translucent band or fatty acid tail layer (FA) were also measured: glutaraldehyde and urea embedment, LW = 66 Å, PH = 35–38 Å, FA = 31 Å; hydroxyethyl methacrylate glutaraldehyde and urea embedment, LW = 55 Å, PH = 18 Å, FA = 37 Å; Epon 812 resin polar dehydrant, LW = 66 Å, PH = 35–38 Å, FA = 31 Å; hydroxypropyl methacrylate polar dehydrant, semihomogeneous matrix without laminations. There was no interlamellar space, so the lamellar width was also the periodicity. In slightly extracted material the PH layer was usually the site of extraction. These results were considered in light of the previously reported measurements of extracted, lipid retained and frozen etched material. In vivo lamellae are probaly 66 Å wide, composed of 35–38 Å PH and 31 Å FA layers and do not exhibit an interlamellar space. Lamellae were observed to fracture at the FA layer at bends, to bifurcate and to anastamose.