Quantitative analysis of the mechanism of negative staining with native collagen fibrils and polar tropomyosin paracrystals

The mechanism of formation of the negatively stained image in electron microscopy was infestigated with native collagen fibrils as a model. The negatively stained image was simulated from the primary structure by using the values of volume or bulkiness of each amino acid residue as a parameter for stain-excluding capacity. The pattern simulated from the bulkiness values gave an excellent fit with the negatively stained image. Since some contribution of positive staining components to negative staining has been suggested, positive staining with uranyl acetate was tested with various washing solutions of different pH. While acidic conditions did not produce any stained image, a positively stained image was easily obtained at alkaline pH. On the other hand, negatively stained images with stains of different charge character remained essentially the same as those obtained with acidic uranyl stains. It was concluded that the contribution of positive components to the negatively stained image is negligible under the conventional conditions for negative staining with uranyl acetate. In order to demonstrate the utility of the analytical method employing the values of "bulkiness," we studied the unknown molecular packing in the polar lead paracrystal of rabbit skeletal tropomyosin. Utilizing the primary sequence data for alpha-tropomyosin we successfully showed the polar paracrystal to be an array of molecules which are parallel and in register. Further, our analysis made it possible to deduce the position of a given residue in the negatively stained pattern of the polar paracrystal.     

Simultaneous demonstration of glycogen and protein in glycosomes of cardiac tissue

Cardiac conduction fibers fixed either in glutaraldehyde and OsO4 or treated additionally en bloc with uranyl acetate were studied in order to demonstrate the structure of glycosomes (protein-glycogen complex). Sections were stained histochemically by periodic acid-thiosemicarbazide-silver proteinate (PA--TSC--SP) for glycogen followed by uranyl acetate and lead citrate (U-Pb) for protein. In control sections periodic acid was replaced by hydrogen peroxide (H2O2). Glycogen appeared in all sections stained by PA-TSC-SP. Protein was poorly contrasted in periodic acid treated histochemical sections taken from fixed in glutaraldehyde and OsO4. Simultaneous staining of glycogen and protein was achieved in sections of tissue treated en bloc with uranyl acetate. This treatment revealed two classes of glycosomes: 1) glycosomes deposited freely in the cytoplasm whose structure was disintegrated after treatment with uranyl acetate: 2) glycosomes associated with other cellular structures that remained intact. Staining of glycogen and protein in the same section demonstrated for the first time the structure of intact glycosomes.     

INTRAMITOCHONDRIAL FIBERS WITH DNA CHARACTERISTICS : I. Fixation and Electron Staining Reactions

Chick embryo mitochondria, studied with the electron microscope, show crista-free areas of low electron opacity. These areas are observable after fixation with osmium tetroxide, calcium permanganate, potassium permanganate, formaldehyde, acrolein, acrolein followed by osmium tetroxide, uranyl acetate followed by calcium permanganate, and acetic acid-alcohol. Staining of sections with lead hydroxide or uranyl acetate, or with both, resulted in an increased density of a fibrous material within these areas. The appearance of the fibrous structures varied with the fixative employed; after fixation with osmium tetroxide the material was clumped and bar-like (up to 400 A in diameter), whereas after treatment of osmium tetroxide-fixed tissues with uranyl acetate before dehydration the fibrous structures could be visualized as 15 to 30 A fibrils. Treatment with ethylenediaminetetraacetate (EDTA) in place of uranyl acetate coarsened the mitochondrial fibrils. After fixation with calcium permanganate or potassium permanganate, or a double fixation by uranyl acetate followed by calcium permanganate, the fibers appeared to have a pattern and ultrastructure similar to that observed after the osmium tetroxide-uranyl acetate technique, except that some of them had a slightly greater diameter (up to 50 A). Other fixatives did not preserve the fibers so well. The fibers appeared strongly clumped by formaldehyde fixation, and were difficult to identify after fixation with acrolein or acetic acid-alcohol. The staining of nucleic acid-containing structures by uranyl acetate and lead hydroxide was improved by treatment of osmium tetroxide-fixed sections with hydrogen peroxide, and the mitochondrial fibers also had an increased density in the electron beam after this procedure. The staining characteristics suggest the fibrous material of chick embryo mitochondria to be a nucleic acid-containing structure, and its variable appearance after different fixations parallels that previously reported, or described in this paper, for the nucleoplasm of bacteria and blue-green algae. The results, in addition to those described in the accompanying communication, indicate that these mitochondria contain DNA.     

PRESERVATION OF MYELIN LAMELLAR STRUCTURE IN THE ABSENCE OF LIPID : A Correlated Chemical and Morphological Study

The fine structure of myelin was studied in glutaraldehyde-fixed rat sciatic nerves depleted of lipid by acetone, chloroform:methanol (2:1 v/v), and chloroform:methanol:concentrated HCl (200:100:1, v/v/v). One portion of each of these nerves, plus the extracts, was saponified and analyzed by gas-liquid chromatography for fatty acids. The remainder of each nerve was stained in osmium tetroxide in CCl₄ (5g/100cc) and was embedded in Epon 812. Thin sections, examined in the electron microscope, revealed the preservation of myelin lamellar structure with a 170 A periodicity in nerves depleted of 98% of their lipids. Preservation of myelin lamellar structure depended on glutaraldehyde fixation and the introduction of osmium tetroxide in a nonpolar vehicle (CCl₄) after the lipids had been extracted. It is concluded that the periodic lamellar structure in electron micrographs of myelin depleted of lipid results from the complexing of osmium tetroxide, plus uranyl and lead stains, with protein.     

A comparative negative staining study of aqueous suspensions of sphingomyelin

Bovine brain sphingomyelin liposomes have been studied by TEM when negatively stained at 4, 22 and 60δC with uranyl acetate,sodium phosphotungstate and amonium molybdate. The liposomes images vary slightly in the different negative stains, yet overall agreement exists once the different permeability and ionic propreties of the stains and the orietation of the liposomes are taken into account. Sodium phosphotungstate possesses an undesirable aggregative action on sphingomylin liposomes, but no aggregation has been encountered with the other stains. The liposomes images at 4 and 22δC are very similar, since both temperatures are beneath the crystalline-liqiud crystalline phase transition temperature (Tc). At 60δC, wich is above the Tc for sphingomyelin, the liposomal particles appear to be much more flexible and accordingly present a more varied shape than at the lower temperatured. The overall conformation of the sphingomyelin liposome is thought to be a ca 50 nm flattened single bilayer vesicle. Nevertheless, data are presented which suggest that some single bilayer disc-like micelles of sphingomyelin are also present.The larger multi-lamellar particular structures or myelion bodies, which are present when solid sphingomyelin is simply disperesed in water, have been studied by negative staining at 22 and 60δC. At 22δC, sphingomylin myelin bodies contain bilayers which exhibit the 26.5 nm undulatory P⨿β′pre-transition phase. The periodic feature is revealed particularly clearly by uranyl acetate. Considerable image complexity is usually present, because of overlapping information from more than one bilayer. Myelin bodies are occasionally split open during the negative staining and the single bilayer regions then reveal the image periodicity with superior clarity. Ammonium molybdate reveals the Pβ′ pre-transition phase undulations rather faintly owing to the permeation of this across the phospholipid bilayers. Sodium phosphotungstate has not been found to reveal this structural feature of the phospholipid bilayer. Ar 22δC some liposomes from spontaneously from the larger lipid bodies, but at 60δC their vesicularization occurs very much more rapidly.     

Morphological aspects of type II alveolar pneumocytes following treatment with puromycin in vivo

Ultrastructural modifications of type II pneumocytes (PNM-II) in mice were analysed 125 and 155 minutes after puromycin treatment (12 mg/100 gm at 0, 30, 60 and 90 minutes). A quantitative evaluation of the cell compartments was carried out and the inhibition of protein synthesis in PNM-II was monitored by light microscopic radioautography, following 3H-leucine injection. In electron micrographs, following a 125-minute puromycin treatment, the number and size of lamellar bodies, the precursors of lung surfactant material appeared markedly reduced. The multivesicular bodies (MVB), which are normally very frequent in PNM-II, had almost completely disappeared, as had composite bodies. Golgi saccules were dilated, while the area occupied by Golgi vesicles was enlarged. Observations following the 155-minute puromycin treatment showed a strong enhancement of these modifications. Smooth and coated vesicles of the Golgi area, as well as peroxisomes, did not appear modified by puromycin. Elongated zones of autophagy were more prevalent after 125-minute treatment than after the 155-minute one. Small bodies were frequently observed in the cytoplasm, near the Golgi zone. They were bounded by a smooth membrane and contained tiny vesicles and/or electron-dense lamellae similar to those present within the lamellar bodies. Parallel membranes formed folds, some of them in continuity with lamellar bodies, thus encircling portions of cytoplasm. These structures, which were few in number in controls, were very frequently observed in treated cells, mainly after the 125-minute treatment. These extensive alterations of PNM-II morphology appeared to be related to a disturbed production of pulmonary surfactant.     

The alveolar-lining layer in the lung of the axolotl,Ambystoma mexicanum

Summary Lungs of neotenic larvae of Ambystoma mexicanum were prepared for maintaining the air-tissue boundary during aldehyde fixation. Four methods of postfixation were applied: 1) osmium tetroxide followed by en-bloc staining with uranyl acetate and phosphotungstic acid, 2) ruthenium redosmium tetroxide, 3) osmium tetroxide-ferrocyanide, and 4) tannic acidosmium tetroxide.Three types of cells line the inner surface of the axolotl lung: 1) pneumocytes, covering the capillaries with flat cellular extensions and containing two types of granules: the osmiophilic lamellar bodies, precursors of extracellular membranous material, and apical granules of unknown significance; 2) ciliated cells, also containing osmiophilic lamellar bodies; and 3) goblet cells filled with secretory granules as well as osmiophilic bodies.The extracellular material forms membranous whorls as well as tubular myelin figures, consisting of membranous backbones combined with an intensely stained substance. This material strikingly resembles the surfactant of amphibian lungs.     

PRESERVATION OF THE ULTRASTRUCTURE OF BACILLUS SUBTILIS BY CHEMICAL FIXATION AS VERIFIED BY FREEZE-ETCHING

The present study on the ultrastructure of Bacillus subtilis was undertaken in order to examine by means of the freeze-etching technique possible structural changes occurring during the chemical fixation procedure (Ryter-Kellenberger (R-K) fixation). Three stages were followed by freeze-etching, viz.: (a) fixation in osmium tetroxide, (b) fixation in osmium tetroxide and posttreatment with uranyl acetate, and (c) fixation in osmium tetroxide, posttreatment in uranyl acetate, and dehydration in a graded series of acetone. Preparations were made after each stage in the presence of 20% glycerol. Good preservation of ultrastructure was observed, after any of the three treatments, of the outer surface of the plasma membrane, and the inner surface of the plasma membrane. No alteration in fracturing properties could be observed. However, if we are to judge by the results of freeze-etching, any of the successive steps of the chemical fixation procedure achieve strong contrast between the nucleoplasmic region and the cytoplasm. Dependent on the quality of fixation, very delicately preserved DNA fibrils or strongly aggregated ones were seen. It appears that R-K fixation is capable of producing more or less distinctly visible changes in the native state of the nucleoplasm in young cells of B. subtilis.     

The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes

The preservation for electron microscopy of saturated phospholipids in general, and phosphatidyl choline (PC)in particular, remains and unsolved problem since OsO(4) and glutaraldehyde are incapable of interacting with PC directly. However, by introducing tannic acid preceding osmication, we were able to demonstrate highly ordered, preserved lamellar structures in model experiments with saturated PC, and in vivo experiments type II pneumocytes of lung tissue. The secretory bodies of the latter are known to contain a high proportion of these saturated phospholipids. In both cases, the repeating periodicity approximated 45 A. It was determined that tannic acid interacts with the choline component of PC to form a "complex," which then could be stabilized by treatment with OsO(4). In the absence of osmication, the PC-tannic acid complex acid did not survive conventional dehydration techniques, but osmication permitted conventional Epon embedment. Sphingomyelin (SPH), which contains choline, behaved similarly in model experiments. But there was no evidence of a comparable reaction with tannic acid using phosphatidyl ethanolamine (PEA), phosphatidyl serine (PS), or phosphstidy inositol (PI). Chemical studies indicted a high pH dependency for the formation of the PC- tannic acid complex. Also, experiments demonstrated its dissociation in various organic solvents. Sharp delineation and great contrast of the polar zones in the ordered lamellar structures was achieved by additional staining with lead citrate thus leading to the conclusion that tannic acid serves as a multivalent agent, capable of simultaneous interaction with saturated PC, OsO(4), and lead citrate stains.     

PREFERENTIAL STAINING OF NUCLEIC ACID-CONTAINING STRUCTURES FOR ELECTRON MICROSCOPY

Oriented fibres of extracted nucleohistone were employed as test material in a study of satisfactory fixation, embedding, and staining methods for structures containing a high proportion of nucleic acid. Fixation in buffered osmium tetroxide solution at pH 6, containing 10^-2 M Ca⁺⁺, and embedding in Araldite enabled sections of the fibres to be cut in which the orientation was well preserved. These could be strongly stained in 2 per cent aqueous uranyl acetate, and showed considerable fine structure. Certain regions in the nuclei of whole thymus tissue could also be strongly stained by the same procedure, and were identical with the regions stained by the Feulgen procedure in adjacent sections. Moreover, purified DNA was found to take up almost its own dry weight of uranyl acetate from 2 per cent aqueous solution. Strongest staining of whole tissue was obtained with very short fixation times-5 minutes or so at 0°C. Particularly intense staining was obtained when such tissue stained in uranyl acetate was further stained with lead hydroxide. Although the patterns of staining by lead hydroxide alone and by uranyl acetate were similar in tissues fixed for longer times (½ hour to 2 hours, at 0°C or 20°C), in briefly fixed material the DNA-containing regions appeared relatively unstained by lead hydroxide alone, whilst often there was appreciable staining of RNA-containing structures. Observations on the staining of some viruses by similar techniques are also described.     

Ultrastructural staining with sodium metaperiodate and sodium borohydride.

This article describes new ultrastructural staining methods for osmicated tissues based on the incubation of sections with sodium metaperiodate and sodium borohydride solutions before uranyl/lead staining. Sections incubated with sodium metaperiodate and sodium borohydride, treated with Triton X-100, and stained with ethanolic uranyl acetate/lead citrate showed a good contrast for the nucleolus and the interchromatin region, whereas the chromatin masses were bleached. Chromatin bleaching depended on the incubation with these oxidizing (metaperiodate) and reducing (borohydride) agents. Other factors that influenced the staining of the chromatin masses were the en bloc staining with uranyl acetate, the incubation of sections with Triton X-100, and the staining with aqueous or ethanolic uranyl acetate. The combination of these factors on sections treated with metaperiodate/borohydride provided a different appearance to the chromatin, from bleached to highly contrasted. Most cytoplasmic organelles showed a similar appearance with these procedures than with conventional uranyl/lead staining. However, when sections were incubated with metaperiodate/borohydride and Triton X-100 before uranyl/lead staining, the collagen fibers, and the glycocalix and zymogen granules of pancreatic acinar cells, appeared bleached. The possible combination of these methods with the immunolocalization of the amino acid taurine was also analyzed. (J Histochem Cytochem 50:11-19, 2002)     

Improved lung preservation relates to an increase in tubular myelin-associated surfactant protein A

Background

Declining levels of surfactant protein A (SP-A) after lung transplantation are suggested to indicate progression of ischemia/reperfusion (IR) injury. We hypothesized that the previously described preservation-dependent improvement of alveolar surfactant integrity after IR was associated with alterations in intraalveolar SP-A levels.

Methods

Using immuno electron microscopy and design-based stereology, amount and distribution of SP-A, and of intracellular surfactant phospholipids (lamellar bodies) as well as infiltration by polymorphonuclear leukocytes (PMNs) and alveolar macrophages were evaluated in rat lungs after IR and preservation with EuroCollins or Celsior.

Results

After IR, labelling of tubular myelin for intraalveolar SP-A was significantly increased. In lungs preserved with EuroCollins, the total amount of intracellular surfactant phospholipid was reduced, and infiltration by PMNs and alveolar macrophages was significantly increased. With Celsior no changes in infiltration or intracellular surfactant phospholipid amount occurred. Here, an increase in the number of lamellar bodies per cell was associated with a shift towards smaller lamellar bodies. This accounts for preservation-dependent changes in the balance between surfactant phospholipid secretion and synthesis as well as in inflammatory cell infiltration.

Conclusion

We suggest that enhanced release of surfactant phospholipids and SP-A represents an early protective response that compensates in part for the inactivation of intraalveolar surfactant in the early phase of IR injury. This beneficial effect can be supported by adequate lung preservation, as e.g. with Celsior, maintaining surfactant integrity and reducing inflammation, either directly (via antioxidants) or indirectly (via improved surfactant integrity).     

A sensitive staining method for detecting acidic polysaccharides in cellulose acetate and agarose gels

A sensitive staining method has been developed for the detection of acidic polysaccharides in cellulose acetate and agarose gels. The method is based on the precipitation of bovine serum albumin by acidic polysaccharides at acidic pH values and the subsequent staining of precipitated protein with amido black or Coomassie brilliant blue R-250 stains. The detection limit of acidic polysaccharides is 15-40 ng on cellulose acetate strips and 50-150 ng on agarose plates. The sensitivity of the described staining technique is of the same order for a wide range of acidic polysaccharides of different origin in contrast to Alcian blue and toluidine blue stains, which detect only mucopolysaccharides of animal origin at comparable levels. The method was also applied to the colorimetric quantitative determination of acidic polysaccharides after electrophoretic separation.     

Refractive index of uranyl-treated bacterial cytoplasm as related to ribonucleic-acid content and growth rate

After fixation and treatment with uranyl acetate solutions, the refractive index (n _D) of the cytoplasm ofCorynebacterium bovis varies with substrate-dependent growth rate and RNA content. The effect, which presumably is due to quantitative binding of uranyl ions by RNA, permits a measurement of the growth rate of single cells in a single-species system by interference microscopy. Temperature-induced changes in growth rate are not reflected in changes ofn _D or RNA content.     

HYDROXYPROPYL METHACRYLATE, A NEW WATER-MISCIBLE EMBEDDING MEDIUM FOR ELECTRON MICROSCOPY

Aldehyde-fixed rat tissues were variously dehydrated and impregnated in water-miscible 2-hydroxypropyl methacrylate (HPMA) containing 3 to 20 per cent water and 0.1 per cent α,α-azobisisobutyronitrile as catalyst for subsequent polymerization with ultraviolet light. Heat polymerization was also effective. Blocks of embedded tissue readily gave ultrathin sections, which required staining by uranyl acetate and/or lead stains to give adequate contrast for electron microscopy. The ultrastructure of pancreas, kidney, muscle, and intestine was well preserved by aldehyde fixation alone. Use of postfixation in osmium tetroxide or direct osmium tetroxide fixation was unsatisfactory. The fine structure of aldehyde-fixed liver from fasted rats was well preserved, whereas that from normal rats showed considerable disorganization and collapse, apparently because of extraction of glycogen during the embedding procedure. Enzymatic extraction of proteins by pepsin and of ribonucleic acid by ribonuclease after either formaldehyde or glutaraldehyde fixation was rapidly effected by direct treatment of ultrathin sections with solutions of the enzymes. In contrast, no digestion of chromatin by deoxyribonuclease could be detected. In spite of this present limitation, HPMA appears to have several advantages over earlier water-miscible embedding media for electron microscopy and to be particularly suitable for ultrastructural cytochemistry.     

The fine structure of the rat pineal and the influence of anti-androgens and castroation]

The pineal body, along with the hypothalamus-hypophysis system, is in the centre of sexual hormone regulation and, in addition to other functions, develops an antigonadotropic action through its organ specific hormone (melatonin). In order to clarify further open questions and to analyse more closely the morphology of the fine structure of the organ, light and electron microscopic studies were made of the pineal bodies of sexually mature male rats after hormonal castration by the administration of cyproterone and cyproterone acetate. The findings were compared with results obtained in the pineal bodies of surgically castrated animals of the same strain. Epiphysectomy was performed 3, 6, 9 and 12 weeks after castration or application of antiandrogen. In the pineal bodies, "light" and "dark" pineal cells and an interstitial cell form could be detected electronmicroscopically. The interstitial cells are found localised near the vessels in particular; their ramifications reach into the perivascular cleft. The light pineal cells preponderate and react essentially in the series; they are therefore considered as the really active form of parenchyma cells. Increased cell activity is already observed three weeks after treatment with antiandrogen: the nucleoli are enlarged, the ribosomes, the mitochondria and ergastoplasm are increased, the endoplasmic reticulum quantified and extended, and also the Golgi regions. The cells are consequently enlarged. Lysosomes also appear which frequently enter the liposomes. The changes in the liposomes after application of antiandrogen are remarkable. Initially they are evacuated, partially drawn out. Later the liposomes are enlarged and increased and often fill the cell body. These pineocytes form an appendage to the castration cells of the hypophysis. The liposomes are in a very close spatial, formal genetic relationship to the Golgi apparatus and to the rough walled reticulum. The larger liposomes apparently arise also through the confluence of smaller ones. Three structural elements of the liposomes could be indentifed: a homogenous, a lamellar and a granular component. The fine morphological reactions are most marked after cyproterone acetate. For the first time, bundles of "microtubuli" are described, the significance of which is not yet clear. They probably arise from the endoplasmic reticulum, they are only found after cyproterone acetate and are presumably due to the gestagen component of the cyproterone acetate. These structures have not previously been observed, either in pineal bodies or in other organs. The structures found after antiandrogen are not so outstandingly recognisable after surgical castration. The biological differences of the surgical compared with hormonal castration therefore seem to be reflected in the cell picture of the pineocytes. Consequently, the pineal body, after treatment with antiandrogen, shows cytostructural changes similar to those of increased anabolism...     

Lung lamellar bodies maintain an acidic internal pH

The internal pH of lung lamellar bodies was investigated with membrane permeable basic amines. Isolated granular pneumocytes and isolated lung lamellar bodies exhibited fluorescence when exposed to 8 microM quinacrine, suggesting accumulation of this dye due to an acidic internal pH. Uptake of [14C]methylamine by isolated lung lamellar bodies was measured to quantitate the intralamellar body pH. In KCl-ATP (10 mM) medium (pH 7.0), the accumulation ratio of methylamine (inside/outside) during 2-min incubation was 8.1 +/- 0.47 (mean +/- S.E., n = 8) indicating an internal pH of 6.1. Lamellar bodies accumulated methylamine almost 30-fold in K+-free mannitol medium indicating an internal pH of 5.6. The pH gradient across the lamellar body membrane decreased when external pH was decreased or when ATP was omitted. The pH gradient was also decreased by the addition of 10 mM NH4Cl, 2 micrograms/ml nigericin, 0.02 mM N,N'-dicyclohexylcarbodiimide, or 1 mM N'-ethylmaleimide. These observations indicate that lamellar bodies maintain an acidic interior (pH 6.1 or below) which is generated by an energy-dependent process.     

A STUDY OF NEWT MITOTIC CHROMOSOMES BY NEGATIVE STAINING

A method is described for bursting single, selected mitotic cells on a fluid surface. Cells from cultures of newt heart tissue were burst on dilute solutions containing potassium and sodium with and without added calcium and also on dilute calcium chloride solution. The material was negatively stained with uranyl acetate or sometimes with ammonium molybdate or sodium phosphotungstate. The bodies of chromatids spread on NaCl/KCl solutions showed many parallel fibers about 150 A in diameter. Loops with a complex nodular structure were observed projecting from the sides and ends of chromatids. In calcium-containing solutions there was evidence of fiber coagulation; the chromatid body was more compact and laterally projecting fibers tended to be pulled out straight. Especially in the absence of calcium the chromosomal fibers had a nodular form and appeared to be composed of irregularly folded fibrillar elements. The question as to whether chromosomal fibers, which range in diameter from about 50 to 300 A, consist of single, folded threads or of two or more adjacent subunits is discussed.     

Ultrastructure of sea urchin calcified tissues after high-pressure freezing and freeze substitution

The improvements brought by high-pressure freezing/freeze substitution fixation methods to the ultrastructural preservation of echinoderm mineralized tissues are investigated in developing pedicellariae and teeth of the echinoid Paracentrotus lividus. Three freeze substitution (FS) protocols were tested: one in the presence of osmium tetroxide, one in the presence of uranyl acetate, and the last in the presence of gallic acid. FS in the presence of osmium tetroxide significantly improved cell ultrastructure preservation and should especially be used for ultrastructural studies involving vesicles and the Golgi apparatus. With all protocols, multivesicular bodies, suggested to contain Ca(2+), were evident for the first time in skeleton-forming cells. FS in the presence of gallic acid allowed us to confirm the structured and insoluble character of a part of the organic matrix of mineralization in the calcification sites of the tooth, an observation which modifies the current understanding of biomineralization control in echinoderms.     

Ultrastructural analysis of the lining layer of the rat pulmonary alveolus.

Using electron microscopy we have examined the lining layer of the rat pulmonary alveolus. This layer appears as a morphological entity 1-3 days after birth: it is composed at first of a filamentous Ruthenium Red-negative material derived from lamellar bodies, and subsequently (4 days after birth) of a homogeneous Ruthenium Red-positive material. This latter material, which corresponds to the epithelia lining of the alveolus typical of adult rats, is presumably derived from a mixture of the filamentous material produced by the lamellar bodies, and a material produced by the alveolar cells 4 days after birth which contains acidic groups which bind Ruthenium Red.