Adenosinetriphosphatase and 5-Nucleotidase Activities in the Plasma Membrane of Liver Cells as Revealed by Electron Microscopy

The sites of reaction product resulting from ATPase and 5-nucleotidase activities remaining in parenchymatous cells of osmium-fixed rat liver were studied by electron microscopy of thin sections. These indicate that both ATPase and 5-nucleotidase activities are localized in the plasma membrane where it folds to form the microvilli of the bile canaliculus, and that 5-nucleotidase activity is also present in the microvilli at the sinusoidal aspects of the cells. It is suggested that these enzymes, particularly ATPase, may play a role in molecular transport or in some kind of membrane activity at the cell surface. Of special interest is the apparent differential localization of these enzymes at the absorptive and secretory regions of the plasma membrane of the cell. It may be of interest to study changes in these enzyme localizations in pathologic states, as a sign of changed cell function. Some of the difficulties in the interpretation of enzyme reaction products seen in electron micrographs are discussed.     

A CYTOCHEMICAL LOCALIZATION OF REDUCTIVE SITES IN A GRAM-POSITIVE BACTERIUM : Tellurite Reduction in Bacillus subtilis

In bacteria the exact location of a respiratory enzyme system comparable to that of the mitochondria of other cells has remained uncertain. On the one hand, the existence of particulate "bacterial mitochondria" has been advocated (Mudd); on the other hand, important enzymes of the respiratory chain were recovered in the cytoplasmic membranes associated with some granular material (Weibull). In order to gain insight into this question, sites of reducing activity were localized in thin sections of bacteria using the reduction of potassium tellurite as an indicator. When this salt was added to the culture medium of Bacillus subtilis, it turned out that in this Gram-positive organism the reduced product is strictly bound at two sites, and that the plasma membrane does not materially gain in electron opacity through deposition of the reduced product. The reduction product is found on or in the membranes of particular organelles, which may possibly be regarded as the mitochondrial equivalents in Gram-positive bacteria, and which are sometimes seen connected to the plasma membrane. The second location is in thin rod-like elements at the cell periphery, possibly the sites from which the flagella emerge.     

A Cytochenfical Study of Acid Phosphatas in the Mycorrhizal Cells of Gastrodia elata Seedling

A cytochemical study has been made to examine the activity of acid β-glycerophosphatase in the mycorrhizal cells of the seedling of Gastrodia elata BI. using thin sectioning technique in which sections were embedded in glycol mathacrylate (GMA). After the seedling was invaded by the hyphae of Mycena osmundicola Lange, two different kinds of infected cells were formed in its root cortex.the outer 1–2 cell layers namely the hyphae-containing cells (or host cells) contained many coiled hyphae pelotons; the inner comparativly large cell layer or fungus-digesting cells contained a few straight hyphae. Localization of acid phosphatase in hyphae-containing cells showed that only a few senescent hyphae retained the enzyme activity and the plant cells did not release hydrolytic enzyme. So it is considered that the hyphal lysis in hyphae-containing cell may be due to autolysis. In contrast, higher acid phosphatase activity was visualized in many vesicles and small vacuoles of the fungus-digesting cells. When a hypha entered a fungus-digesting cell through a hyphae-containing cell, a number of enzyme granules (i. e, enzymecontaining vesicles) gathered around it. Later on the enzyme granules expanded gradually and became small enzyme vacuoles of 1.6–2.0 μm in diameter. Still later the small enzyme vacuoles fused with each other to form a large vacuole in which a part of an invading hypha was enclosed and gradually digested by hydrolytic enzymes. Finally,the digesting vacuole changed into a residual body containing some metabolic waste. The above results suggest that fungus-digesting cells can actively release hydrolytic enzymes by lysosomal vesicles to digest the invading hyphae, but such function is not present in the hyphae-containing cells,the role of which may be attributed to attracting and controling the invading hyphae.     

Fine structural and histochemical studies on salivary glands of Peripatoides novae-zealandiae (Onychophora) with special reference to acid phosphatase distribution

The Onychophora feed on small arthropods and produce saliva when ingesting prey. Although saliva undoubtedly helps to liquefy the food its constituents have not yet been fully described. The salivary glands, two long tubes of glandular epithelium, are known to secrete a powerful protease, however, besides other enzymes and mucus. In Peripatoides novae-zealandiae there are protein-secreting cells of three types, referred to here as columnar, cuboidal and modified cells, and mucus cells. The anterior two-thirds of the gland show most cell diversity, while the posterior region consists mainly of columnar cells. These are the most numerous elements overall and they probably secrete salivary protease. In thick resin sections the granules of all protein-secreting cells stain strongly with methylene blue. Those of columnar cells are markedly uneven in size and accumulate distally, eventually filling the cytoplasm. More proximal Golgi regions may be discernible. Mucus cells are all of one type and their secretion droplets are stained lightly by methylene blue. The electron microscope shows that distal microvilli, desmosomes and septate junctions are common to all gland cells. In columnar cells, secretory material is contributed by Golgi complexes and by rough endoplasmic reticulum. Early secretory vacuoles containing dense material are seen in the concavity of Golgi regions. They are precursors to larger condensing vacuoles whose contents have a more flocculent appearance, and which may attain 3–4 μm in diameter. These evolve into secretory granules, usually of uneven texture, which are up to 2–5 μm in diameter. Histochemical tests for acid phosphatase show moderate amounts of enzyme throughout the gland. In whole mounts and sections the strongest reaction is in a band of cuboidal cells along the anterior median border. Columnar cells show a diffuse cytoplasmic reaction towards the base and sometimes distal to the nucleus, and mucus cells may also react strongly round the nucleus. Cytoplasm near the lumen shows little reaction. The secretory granules do not appear to contain active enzyme. Under the electron microscope a positive reaction for acid phosphatase is seen in lysosomal derivatives near the base and lateral periphery of gland cells. These bodies are probably autophagic vacuoles and they may contain membranous whorls and possibly old secretion granules. Acid phosphatase is involved also in the elaboration of new secretory granules in both columnar and mucus cells. Dense reaction product is found in a system of interconnected tubules and cisternae near the innermost face of the Golgi complex, which is interpreted as GERL. Acid phosphatase is present in the peripheral zone of adjacent early secretory vacuoles, and interconnections occur between GERL and secretory vacuoles. It is suggested that GERL tubules containing the enzyme may fuse with early secretory vacuoles and release acid phosphatase at their periphery. The acid phosphatase reaction is negative in large condensing vacuoles and most secretory granules. These findings are consistent with what is known from mammalian cells, including those of salivary glands.     

Tritrichomonas foetus: ultrastructure and cytochemistry of endocytosis

Tritrichomonas foetus ingests horseradish peroxidase, native ferritin, cationized ferritin, and 0.08 micron latex beads by a process which involves the formation of pinocytic vesicles. These vesicles fuse with each other and with lysosomes forming large vacuoles. Biochemical determinations on the ingestion of horseradish peroxidase and morphometric analysis on the ingestion of cationized ferritin covered latex beads indicated that T. foetus has high endocytic activity. The process of ingestion of the various tracers used was analyzed by transmission electron microscopy of thin sections and freeze fracture replicas.     

Structure of mitochondria and vacuoles of Candida utilis and Schizosaccharomyces pombe studied by electron microscopy of serial thin sections and model building

The structure of mitochondria and of vacuoles in Candida utilis and Schizosaccharomyces pombe has been studied by electron microscopy of serial thin sections and subsequent model building. The models of the two cells of C. utilis which were studied confirmed our earlier findings, made by high voltage electron microscopy of thick sections, that there is a single, branched and continuous mitochondrial network in the cell (Davison & Garland, 1975). A model of a S. pombe cell showed that the mitochondrial structure was far more continuous than expected from inspection of thin sections, there being but two large and two small mitochondria. The models demonstrated that the few large vacuoles in C. utilis were interconnected into a single cluster, whereas in S. pombe there were two separate complexes of interconnected vacuoles towards each pole of the cell.     

THE ULTRASTRUCTURE AND HISTOPHYSIOLOGY OF HUMAN ECCRINE SWEAT GLANDS

The electron microscopy of human eccrine sweat glands has been studied before and after stimulation by pilocarpine iontophoresis. The identity of the dark and clear cells in the secretory segment as defined by Montagna et al. (23) was determined by studying serial sections, thin for electron microscopy and thick for light microscopy. Cells with numerous apical secretory vacuoles are termed mucoid (dark) cells, since these vacuoles stain positively for acid mucopolysaccharide. Clear cells are intimately associated with intercellular canaliculi. The "cuticular border" of surface cells of the duct is a condensation of tonofilaments and granules. Numerous mitochondria are concentrated in basal cells of the duct. The presence of mucoid cells in the secretory segment may bear on the interpretation of the pathologic findings in the disease cystic fibrosis of the pancreas, and suggests that this disease may be due to a basic disorder of mucopolysaccharide production. The possible roles of the various cellular components in the elaboration of sweat are discussed.     

A STUDY OF METASTATIC RENAL CALCIFICATION AT THE CELLULAR LEVEL

Experimental metastatic calcification in the proximal convoluted tubules of rat kidney, produced by large doses of vitamin D, has been studied with a variety of techniques. These techniques include the examination of thin sections of Araldite-embedded material under the electron microscope, selected area electron diffraction, and several histochemical methods. Two types of mineral are found in relation to the proximal convoluted tubule. The first form consists of aggregates of elongated crystals within cytoplasmic vacuoles of the proximal tubular cells. The dimensions of these crystals are consistent with those of hydroxyapatite. The other type of mineral deposit is found in and adjacent to the extracellular phase of the basal infoldings of these tubules. The latter deposits are made up of smaller crystals arranged in layers. These crystals could not be definitely identified by means of selected area electron diffraction. The observations are discussed in relation to calcium transport by the proximal convoluted tubule and also in terms of mechanisms of pathological calcification.     

Morphology of the green livers in upstream migrants of Petromyzon marinus L.

A morphological comparison was made of the green livers of male and female lampreys (Petromyzon marinus L.) collected during the upstream (prespawning) migration. Light and electron microscope histochemistry for iron, and both thin sections and freeze-fracture replicas in the electron microscope, revealed some sexual dimorphism in these livers. Ferric iron is much more abundant in the liver of females and is present in the cytoplasmic matrix, in dense bodies, and in vacuoles of hepatocytes. The numerous vacuoles of females may be the deposition site of biliverdin and other bile components that would account for the darker green coloration of the liver compared to males. Hepatocytes in females are also characterized by prominent rough endoplasmic reticulum and Golgi apparatus that reflect the involvement of the cells in vitellogenesis. The presence of numerous lipid droplets in the hepatocytes of males indicates that the liver is an important storage site for fat. The lipid droplets are associated with electron-dense deposits of unknown nature. Large gap junctions typify the parenchymal cells of both male and female livers. Perisinusoidal and sinusoidal cells are similar to those in the nonparenchymal region in other vertebrate livers, namely, endothelial and Kupffer cells, lipocytes (Ito), and some granulated cells. The relationship of lipocytes to fibrous tissue and fibrogenesis is discussed.     

Fine structure and tellurite reduction in azotobacter vinelandii

Summary The fine structure of Azotobacter vinelandii was examined using a micro-colony embedding method. With this technique the difficulty of obtaining well preserved bacterial flagella in thin sections of material prepared in the usual fashion for electron microscopy was overcome, as the cells and their appendages were held in their natural position. The insertion of flagella and their substructure as revealed by thin sectioning and negative staining was studied. The results obtained on the fine structure of the flagellum is discussed and a possible interpretation of the arrangement of sub-units is presented in a model. Some new inclusions and membranous structures in the cytoplasm of the cells are described. These structures do not appear to be involved in tellurite reduction. These is no evidence to indicate that the flagellar insertion sites showed any activity of tellurite reduction. Thus in Azotobacter, other systems seem to be responsible for the ability of the cells to reduce tellurite.     

ULTRASTRUCTURAL STUDIES PLASMA MEMBRANE RELATED SECONDARY VACUOLES IN CULTURED CELLS

The plasma membrane of cultured cells of several plant species was observed to possess invaginations, or secondary vacuoles, of variable size in the adjacent cytoplasm. These structures, which occurred in cells at different phases in vacuolation, were very numerous in thin sections of some cells but fewer in others. In vacuolated cells enlarged secondary vacuoles protrude into the primary vacuole but are delimited from the tonoplast by an intermembrane zone of variable width. The plasma membrane at the orifice of an invagination may fuse and detach the secondary vacuole from the membrane to form in the cytoplasm a structure bounded by a single membrane. Complex accumulations of membranes consisting of spherical, tubular, and laminar structures, possibly containing cytoplasm, may develop within secondary vacuoles. Contents of many of these vacuoles arise from folds along its limiting membrane which pinch off into the interior of the secondary vacuole. A fibrous substance, possibly derived from the wall, is present in some secondary vacuoles. Observed folding of the plasma membrane and measurements of membrane width of various organelles and cytomembranes support an interpretation that endocytosis occurs in cultured cells.     

Amphotericin B-induced changes in K+ content, viability, and ultrastructure of yeast-phase Histoplasma capsulatum.

Yeast-phase cells of Histoplasma capsulatum were challenged with amphotericin B, and membrane perturbation was monitored by K+ efflux. Suspensions of washed cells readily absorbed about 1.12 microgram of amphotericin B per mg (dry weight) and further nonspecific sites were also apparent. The dose-response curve for initial rate of K+ efflux was sigmoidal within the range 0.1 to 1.0 microgram of amphotericin B per ml. A fungistatic concentration of amphotericin B (0.3 microgram/ml) evoked an efflux of 85 to 90% K+ from the cells within 15 min, but cell viability decreased only 13% (yeast phase) or 33% (transformed to mycelial units). Ultrastructural changes in treated cells were detected within 5 min, and the hallmark was expansion of vacuoles during the 1-h monitoring period. In contradistinction to a previous report, the appearance of the protoplasmic membrane was not altered by fungistatic concentration. When treated cells were returned to a fresh growth medium, there was a pronounced lag (20 h). During this apparent recovery phase, the large vacuoles fragmented and returned to normal size. It is proposed that vacuoles of H. capsulatum act as a spatial buffer of considerable survival value to stressed cells.     

Topographical and planar distribution of Helix pomatia lectin-binding glycoconjugates in secretory granules and plasma membrane of pancreatic exocrine acinar cells of the rat: demonstration of membrane heterogeneity

The lectin-gold technique was used to detect Helix pomatia lectin (HPL) binding sites directly on thin sections of rat pancreas embedded in Lowicryl K4M and on freeze-fractured preparations of rat pancreas submitted to fracture label. On thin sections of acinar cells, whereas the content of zymogen granules was negative or weakly labeled, the limiting membrane displayed a high degree of labeling. In the Golgi complex, labeling by HPL was localized on the trans saccules and the limiting membrane of the condensing vacuoles. The latter appeared to be more intensely labeled than the membrane of the zymogen granules. Intense labeling by HPL was also observed along the microvilli and the plasma membrane. In contrast to the weak labeling of the zymogen-granule content, labeling of the acinar lumen was intense. Fracture-label preparations revealed preferential partition of HPL-binding sites to the exoplasmic half of the zymogen-granule and plasma membranes. The population of zymogen granules was, however, heterogeneous with respect to labeling intensity; the exoplasmic fracture-face of the plasma membrane was intensely and uniformly labeled, while the protoplasmic membrane halves were only weakly labeled. These observations were further confirmed and extended by the thin-section fracture-label approach. In addition, favorable profiles of thin sections of freeze-fractured zymogen granules showed that the labeling was not associated with the external surface of the limiting membrane, but rather localized over the exoplasmic fracture-face. We conclude that 1) zymogen granules contain little HPL-binding glycoconjugates, 2) HPL-binding sites are preferentially associated with the exoplasmic half of the zymogen-granule and plasma membranes, and 3) the limiting membrane of the immature condensing vacuoles carries a greater number of HPL-binding sites than that of the mature zymogen granules. These last, in turn, constitute a heterogenous population with respect to labeling density. These results support the current view that glycoconjugates are directed toward the lumen in secretory granules but become external to the cell surface after fusion of the secretory-granule membrane with the plasma membrane. Also, the results reflect membrane modifications during the maturation process of secretory granules in the exocrine pancreas in which glycoproteins are removed from the limiting membrane of the granule to become soluble and secreted with the content.     

An evaluation of the form of vacuoles in thin sections and freeze-etch replicas of root tips

Summary A comparison is made of the form of vacuoles in thin sections and freeze-etch replicas of root tips. In sections, vacuoles exhibit a diversity of shapes, the greatest irregularity being found with fixation in aqueous KMnO₄. Vacuoles of frozen-etched roots are mainly spherical. They are not found with narrow extensions or angular irregularities but retain a turgid appearance with a smoothly contoured tonoplast, except in some prefixed and poorly frozen fresh cells. As freeze-etching avoids artifacts of sectioning techniques it is considered that results obtained from freeze-etching give a more accurate picture of the shape of vacuoles. The irregular shapes of vacuoles in thin sections are apparently caused by shrinkage during fixation. When shrinkage is severe, portions of the tonoplast become apposed and superficially resemble profiles of endoplasmic reticulum.     

ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF THIN SECTIONS: THE GOLGI ZONE AS A SITE OF PROTEIN CONCENTRATION IN PANCREATIC ACINAR CELLS

Electron microscopic radioautographs of guinea pig pancreatic exocrine cells were obtained by covering thin sections (~ 600 A) of OsO₄-fixed, methacrylate-embedded tissue with thin layers of Ilford K-5 nuclear research emulsion. After an exposure of 13 days at 4°C., the preparations were photographically processed, stained with uranyl acetate, and examined in an electron microscope. The label used was leucine-H³ injected intravenously 20 minutes before collection of the specimens. Conventional radioautographs of thicker sections (0.4 micron) were also examined in a phase contrast microscope. The advantages obtained from electron microscopic radioautography are: the higher radioautographic resolution (of the order of 0.3 micron) due to the thinness of the emulsion and the specimen, and a high optical resolution permitting a clear identification of the labeled structure. In the guinea pig pancreas this technique demonstrated that, at the time studied, newly synthesized proteins were concentrated in the structures of the Golgi complex and especially in large vacuoles partially filled with a dense material. The vacuoles are probably a precursor to the secretion granules (zymogen granules) in which the label becomes segregated at a later time. These observations demonstrate directly the role of the Golgi complex in the secretion process. They also illustrate the possibilities of this method for radioautography at the intracellular level.     

STUDIES ON THE MODE OF ACTION OF EXCESS OF VITAMIN A : VII. Changes in the Fine Structure of Erythrocytes during Haemolysis by Vitamin A

Rabbit erythrocytes have been haemolysed by treatment with vitamin A alcohol and the sequence of changes in the fine structure of the cells during lysis has been investigated by phase contrast microscopy of intact cells and electron microscopy of thin sections. The initial effect of the vitamin, which occurs within 1 minute, is the production of cells of bizarre appearance which have a greatly increased surface area relative to untreated cells. Large indentations appear in the surfaces of the cells, and vacuoles are formed from the indentations by a process that resembles micropinocytosis. The cells then become spherical and loss of haemoglobin begins as breaks appear in the membranes of some cells; finally, ghosts are produced that are no longer spherical but still contain numerous vacuoles. These observations support the thesis that one site of action of vitamin A is at lipoprotein membranes.     

大鼠睾丸间质细胞的自体吞噬活动

本文结合超微结构和细胞化学观察,研究大鼠睾丸间质细胞(Leydig细胞)中溶酶体的结??构与功能。观察结果表明,大鼠睾丸间质细胞中高尔基体非常发达,在高尔基体的成熟面存在着CMP酶阳性反应的GERL系统,说明这种细胞有不断产生溶酶体的能力。细胞化学结果也证实在睾丸间质细胞有较多的初级和次级溶酶体。睾丸间质细胞不仅有较多的溶酶体,而且还有相当数量的自噬小体,存在着活跃的自体吞噬活动。自噬小体的界膜来源于特化的光面内质网或高尔基体膜囊,包围的内容物主要是光面内质网和少量线粒体。当自噬小体与溶酶体融合后即成为自体吞噬泡,由于酶的消化作用,自体吞噬泡内的细胞器有一系列形态变化。根据CMP酶细胞化学反应,可以区分自噬小体和自体吞噬泡,后者是一种次级溶酶体,呈CMP酶阳性反应。睾丸间质细胞是分泌雄性激素的内分泌细胞,其光面内质网和线粒体在类固醇激素分泌中起重要作用,自体吞噬活动的结果是去除部分内质网和线粒体,可能在细胞水平上起着对雄性激素分泌的调节作用。     

Relationships between membranous organelles in amoebae studied by electron microscopic cytochemical staining

Summary The intracellular location of a variety of enzymes was studied in Amoeba proteus with the use of electron microscopic cytochemical methods, in an attempt to assess the relationships between different membranous organelles. One group of enzymes, including nucleoside diphosphatases (IDPase, UDPase, GDPase, ADPase), carbamoyl phosphatase, alkaline phosphatase, and BAXD oxidase was localized mainly in the rough endoplasmic reticulum, nuclear envelope, and convex side of the Golgi apparatus. Esterase activity had a similar localization except that the Golgi apparatus was "stained" throughout most of its extent. A second group of enzymes was found in Golgi cisternae and vesicles, and in some vacuoles. This group included acid phosphatase, thiamine pyrophosphatase, and aryl sulfatase. Some enzymes previously detected in cytoplasmic membranes of other cells, including glucose-6-phosphatase, showed little or no activity in amoebae. The results suggest that there are chemical similarities and probable functional relationships between the rough endoplasmic reticulum, the nuclear envelope, and the convex side of the Golgi apparatus. On the other hand, the concave pole of the Golgi apparatus, aggregates of smooth tubules and vesicles, and the cell surface appear more closely related to one another than to the endoplasmic reticulum and the convex side of the Golgi apparatus. The cytochemical similarity between the Golgi apparatus and certain vacuoles such as food vacuoles may reflect the role of the Golgi apparatus in the formation of lysosomes. The locations of reaction products of the various enzymes in amoebae are compared with observations reported for other cell types.Supported by a research grant (VC-169) from the American Cancer SocietyThe author is indebted for technical assistance to Mrs. Sue Thompson and Mrs. Christine Folsom-Kovarik     

The distribution of some hydrolytic enzymes in the cells of the digestive gland of certain lamellibranchs and gastropods

Five hydrolytic enzymes (acid phosphatase, aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, and non-specific esterase) have been studied histochemically in the cells of the digestive gland of Mytilus edulis, Helix aspersa , and certain other lamellibranchs and gastropods. All the enzymes studied have basically similar distributions.
In the digestive cells, the enzymes occur in cytoplasmic granules which are believed to be primary lysosomes; in vacuoles which contain phagocytosed food material; and in vacuoles containing lipofuscin granules, which are the residues of digestive activity.
In the basiphil cells of M. edulis , most of the enzymes are localized in a few cytoplasmic granules; non-specific esterase, however, is found throughout the cytoplasm. In the calcium cells of H. aspersa and the other pulmonate gastropods studied, the enzymes are either in cytoplasmic granules, or distributed diffusely throughout the cytoplasm. Acid phosphatase is also found in the calcium spherules, especially in H. aspersa.
In the excretory cells of H. aspersa and the other pulmonates studied, the enzymes are found in granules in the cytoplasm, and in the lipofuscin granules which lie in the vacuoles of these cells.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.