SITES OF CYTOPLASMIC RIBONUCLEOPROTEIN-FILAMENT ASSEMBLY IN RELATION TO HELICAL BODY FORMATION IN AXENIC TROPHOZOITES OF ENTAMOEBA HISTOLYTICA

Axenic trophozoites of Entamoeba histolytica showed increased logarithmic growth but absence of "chromatoid" material (stacked helical arrays of ribonucleoprotein [RNP]) when grown in an all-liquid monophasic culture. Organisms grown in a liquid overlay on a semisolid slant (biphasic medium) showed slow logarithmic growth and the presence of chromatoid material. Chromatoid material accumulated in the rapidly growing trophozoites from monophasic culture during treatment with the Vinca alkaloid, vinblastine. Many of the glycogen-free regions of vinblastine-treated trophozoites as well as, to a lesser degree, of normal cells grown in monophasic and biphasic cultures, contained free ribosomes and randomly oriented 60 A filaments. As ribonucleoprotein assumed the packed helical configuration, areas consisting of parallel, packed filaments could be detected adjacent to and continuous with the ordered RNP arrays. This arrangement could be visualized most frequently in vinblastine-treated trophozoites grown in monophasic cultures. Depending on the tilt of the section with respect to the longitudinal axis of individual helices, 60 A filamentous material could be demonstrated associated with the RNP helices. Localization of ribonucleoprotein precursors was followed by means of high resolution radioautography with uridine-³H and cytidine-³H. With a short (30-min) pulse, label could be visualized only over the glycogen-free areas containing free ribosomes and filaments. With 60-min pulses, label could also be seen over the packed helical arrays. With 30-min pulses followed by a 60-min cold chase, label was seen chiefly over RNP helices. It is postulated that the areas containing ribosomes and filaments represent sites of assembly of the RNP helices possibly on a filament protein column. The possibility that the final helical configuration may be due to a property of this protein is suggested.     

STUDIES ON THE FINE STRUCTURE OF ULTRACENTRIFUGED SPINAL GANGLION CELLS

The following structures were observed in electron micrographs of the mouse spinal ganglion cells: Nissl bodies composed of both aggregated rough-type, largely oriented, membranes of the endoplasmic reticulum and discrete particles; short rodlike mitochondria with well-developed transverse, obliquely or longitudinally arranged cristae, and a relatively typical Golgi complex. The components of ultracentrifuged ganglion cells (400,000 times gravity for 20 minutes) are stratified, the layers appearing in the order of their decreasing density as follows: (1) A microsomal or ergastoplasmic layer which may be further divided into three sublayers without sharp boundaries, namely, a discrete particle layer, a layer of discrete particles and highly distorted membranes of the endoplasmic reticulum, and a layer composed of relatively intact, but stretched membranes of the endoplasmic reticulum and discrete particles. (2) Mitochondria constitute a relatively broad layer. They are sometimes stretched; however, they retain most of their fine structure. The stratified nucleus is found within the mitochondrial layer. (3) A relatively wide layer of tightly packed vesicles. (4) At the centripetal end, resting against the cell membrane, are a few lipid vacuoles. A comparison is made between the ultrastructure of the stratified layers in situ and those described by others in differentially ultracentrifuged homogenates.     

Morphological observations on cultured lampbrush-stage Rana pipiens oocytes.

Early lampbrush-stage oocytes are characterized by small lampbrush chromosome loops, a small amount of ribonucleoprotein (RNP) matrix on the loops, small nucleoli, few RNP particles in the nucleoplasm, and a smooth germinal vesicle contour. In vitro culture of these oocytes in serum-free culture medium for 24 hr at 18°C promotes a number of morphological changes in the oocytes: The lampbrush loops increase in diameter and acquire extensive RNP matrix, the nucleoli increase in size and complexity, the nucleoplasm accumulates numerous polymorphic RNP particles, and the germinal vesicle envelope acquires a sacculated contour. These characteristics are typical of the in vivo maximum lampbrush stage, and their appearance is due to an apparent in vitro acceleration of the lampbrush phase. Two possible interpretations of these observations are discussed.     

The Ultrastructure of Trophozoites of Entamoeba histolytica with Particular Reference to Spherical Arrangements of Osmiophilic Cylindrical Bodies

SYNOPSIS. Trophozoites of Entamoeba histolytica were cultured under xenic, monoxenic and axenic conditions. Some of the monoxenically cultured trophozoites were grown in the presence of Bacteroides symbiosus and others in the presence of Trypanosoma cruzi (Mexican strain). Other trophozoites were obtained from experimentally produced amebic liver abscesses in hamsters. The trophozoites were centrifuged and prepared for study by electron microscopy. Acid phosphatase activity in the parasites was determined by cytochemical reactions.
The various elements of the trophozoites were described and special attention was given to the spherical arrangements of electrons-dense cylindrical units surrounding finely granular material. Their presence was independent of the strain studied and of the nutrient elements in the culture medium. The cylindrical units probably arise from endoplasmic reticulum elements and their possible function in the digestive processes and aggression mechanisms of the trophozoites is discussed.
Acid phosphatase activity was found in round non-branching intranuclear bodies in trophozoites cultured in various media. Whether these bodies represent part of the lysosomal system of the parasite is unknown.
In preliminary work on the action of some amebicidal drugs, a special arrangement of cytomembranes morphologically similar to a Golgi complex was frequently seen in trophozoites of E. histolytica.     

Fine Structural Observations of the Erythrocytic Stages of Plasmodium chabaudi Landau, 1965*

SYNOPSIS. Electron microscopic examination of Plasmodium chabaudi in mouse erythrocytes revealed many characteristics resembling those observed in other mammalian malarial parasites. A double unit membrane surrounds the trophozoite cytoplasm which contains many ribonucleoprotein particles, a limited amount of endoplasmic reticulum and membraned organelles including sausage-shaped vacuoles and multilaminated membraned bodies. More or less circular double membraned vacuoles, possibly cross sections of the sausage-shaped vacuoles, are common. Typical protozoan mitochondria are lacking. The limiting membrane of the merozoites is triple-layered. Paired organelles and small dense bodies are found in the merozoites along with dense granular masses in the nuclei. Trophozoites have cytostomal structures as well as invaginations of the plasma membrane at sites where no cytostomes are evident. Digestion appears to occur in single membrane-bound vesicles which contain one to several pigment grains. P. chabaudi frequently contains multiple food vacuoles and has polymorphism manifested in part by the presence of cytoplasmic extensions and of nuclei with a variety of shapes. Several apparently free forms are noted, often accompanied by a thin rim of host cytoplasm. “Appliqué” forms are common among the trophozoites as are forms in which 2 or more trophozoites are joined together. Finally, alterations in the host cytoplasm resembling the socalled Maurer's clefts are frequent. Ferritin-containing vacuoles also appear in the host cell.     

The use of formaldehyde-treated 131-I-albumin in the study of digestive vacuoles and some properties of these particles from mouse liver

The trichloroacetic acid-soluble radioactivity released during incubation of mouse liver particles containing intravenously injected formaldehyde-treated ¹³¹I-albumin consisted almost entirely of ¹³¹I-iodotyrosine. The material was shown to be excreted into the medium and was not due to disruption of the particles by acid. Triton X-100 or the absence of sucrose in the medium inhibited hydrolysis of the particle-associated labeled protein. This inhibition was due to disruption of the digestive vacuoles and dilution of the protein and cathepsins in the suspending medium. These results and other experimental evidence strongly suggest that the ¹³¹I-albumin-containing liver particles are digestive vacuoles. The results also establish that ¹³¹I-albumin may be used to study these vacuoles. High concentrations of sucrose (1 M) inhibited degradation of intraparticulate protein. However, 1 M salts inhibited only the rate of the digestion. Sucrose had an inhibitory effect on a crude cathepsin preparation, and salts stimulated the activity when ¹³¹I-albumin was used as substrate. The effect of high sucrose concentrations as an inhibitor of protein hydrolysis within digestive vacuoles was, therefore, most likely due principally to an inhibition of cathepsin activity within the vacuoles. The effect of salt was probably caused by a stimulation of both intra- and extra-particulate cathepsin activities, although 0.5–1.0 M KCl appeared to protect the particles.     

Ultrastructure and cytochemistry of glycogen-containing vacuoles in gastrodermal cells in developing hydranths of a hydromedusan coelenterate

Hydranth buds from the colonial hydroid Sertularia pumila (Hydromedusae) were observed by electron microscopy during their development. Before hydranth expansion, the gastrodermal columnar digestive cells had large numbers of vacuoles. These vacuoles contained many membranous components as well as α-glycogen and dense ring- and crescent-shaped bodies. The rings and crescents were not osmiophilic, but did react to periodic acid oxidation in the PA-TSC-SP test for carbohydrate. These structures were digestible by α-amylase and pullulanase. The chemical analyses and the close association of the rings and crescents to α-glycogen particles showed that they may be a highly condensed form of glycogen. Golgi bodies in association with the gastrodermal vacuoles had acid phosphatase activity. This enzyme was only slightly active in the vacuoles. It is suggested that the vacuoles arc primarily storage organelles with a potential for digestion.     

Digestive system membranes: freeze-fracture evidence for differentiation and flow in Paramecium

Freeze-fractured membranes of digestive vacuoles of randomly feeding Paramecium caudatum exhibit dramatic differences in intramembrane particle (IMP) number and distribution on both E- and P-fracture faces. By pulse-feeding latex spheres to cells we have demonstrated that these differences are related to the age of the digestive vacuoles, and that the membranes of such vacuoles undergo a specific sequence of changes during the digestive cycle. Young digestive vacuoles (DV-I; less than or equal to 6 min), nascent vacuoles still connected to the cytopharynx, and discoidal vesicles, from which vacuole membrane is derived, all have a highly particulate E face and a less particulate P face. As early as 3 min after feeding, a second category of digestive vacuoles (DV-II) can be recognized, which are both considerably smaller in diameter and lack particles on their E face. These findings suggest that the endocytic removal of DV-I membrane material associated with the formation of DV-II vacuoles involves a concomitant and selective removal of E-face particles, as essentially no changes are seen in the density of P-face particles on the two types of vacuoles. Beginning at 10 min the first DV-III vacuoles are encountered. These are both larger than the DV-II vacuoles and possess very prominent E-face particles, which resemble those on the E face of the numerous lysosomes bordering the digestive vacuoles. DV-III vacuoles also exhibit a substantial increase in P-face particles. These membrane changes closely parallel, and are probably correlated with, the physiological events occurring within the vacuole lumen: concentration of food, killing of prey, and digestion. Calculations of the amount of membrane removed from DV-I to form DV-II and of the increase in membrane surface area during the transition from DV-II to DV-III indicate that as much as 90% of the initial phagosome (DV-I) membrane can be removed before digestion begins. The enlargment of DV-II must be caused by fusion with adjacent lysosomes which also contribute the new populations of IMPs to the DV- III membrane. The appearance of numerous endocytic structures on older DV-III vacuoles suggests that membrane is retrieved from DV-III before defecation.     

Freeze-etching observations of trophozoites of pathogenic Entamoeba histolytica.

Pathogenic Entamoeba histolytica trophozoites were studied by the freeze-etching (FE) technique of electron microscopy. Surface replicas of intact cell membranes were highly convoluted with numerous invaginations, evaginations, and undulations. Sperical depressions and elevations varying from 0.5 mu to 1.0 mu in diameter were commonly present on the external cell membrane and appeared to represent an extracellular secretory mechanism of trophozoites. Cleaved surfaces of amebae exhibited a granular and lumpy cytoplasm in which there were many vesicles and vacuoles that ranged in diameter from 0.2 mu to 9.0 mu. Some vacuoles contained tightly enveloped bacteria, while others contained bacteria and host cytocomponents. Occasional vesicles and vacuoles appeared to be fused to each other. Replicas of FE nucleus were enclosed by double nuclear membranes which were fenestrated by numerous sperical pores measuring approximately 640 A in diameter and spaced at intervals of 650 A. Counts of nuclear pores were possible and indicated 35 pores per square micron on the nuclear envelope. Golgi apparatus, mitochondria and well formed endoplasmic reticulum were absent in FE replicas. This was in agreement with electron microscope observations on thin sections previously reported by other investigators.     

The elementary RNP fiber — not the higher order structure — determines the all-or-none disintegration behaviour of balbiani ring pre-messenger RNP particles upon RNase A treatment

Summary— Balbiani ring premessenger ribonucleoprotein (RNP) particles are built from a 7-nm RNP fiber which is tightly folded into a ring-shaped RNP ribbon. Isolated particles are known to disintegrate in an all-or-none fashion upon RNase A treatment. In the present study we investigated whether this mode of disintegration is dependent on an intact particle structure or is inherent in the 7-nm fiber. When treated at low ionic strength, the Balbiani ring (BR) particles lost their higher order structure and the 7-nm fiber was unpacked, as evidenced by sucrose gradient sedimentation and electron microscopy. When treated with RNase A, unfolded as well as intact particles disintegrated in the all-or-none fashion, with similar kinetics and without apparent intermediates. Proteinase K treatment, however, obliterated this pattern: the protein-free particle RNA degraded progressively. As the typical disintegration pattern of the particles was not altered by unfolding, but was lost by deproteinization, the all-or-none mode of disintegration is likely to be a property of the 7-nm RNP fiber.     

A family of double helices of alternating poly(gamma-benzyl-D-L-glutamate), a stereochemical model for gramicidin A.

Poly(γ-benzyl-d-l-glutamate) with strict alternation of l and d residues is found to exist, in addition to the α_DL and π_DL^4.4 helical structures already described (Heitz et al., 1975a), in four more helical structures. Models based on double helices made of antiparallel strands are proposed for all four structures, based on infrared, X-ray and electron diffraction data. These double helices are, like the single-stranded π_DL helices, specific to polypeptides with a strict stereosequence of alternating l and d residues. The diameter of the helical core of three of these helices appears to depend on the dimensions of the solvent molecules. Conformational angles (located in the β regions) and atomic co-ordinates determined by conformational energy analysis are given for the four structures. Experimental conditions used to obtain these helices, and to induce transconformations between the various helical structures of PBd-lG are described. The present investigations on PBd-lG help to make more precise the structure and geometry of models proposed (Veatch et al., 1974) for the antibiotic gramicidin A.     

THE FINE STRUCTURE OF ACANTHAMOEBA CASTELLANII : I. The Trophozoite

The fine structure of the trophozoite of Acanthamoeba castellanii (Neff strain) has been studied. Locomotor pseudopods, spikelike "acanthopodia," and microprojections from the cell surface are all formed by hyaline cytoplasm, which excludes formed elements of the cell and contains a fine fibrillar material. Golgi complex, smooth and rough forms of endoplasmic reticulum, digestive vacuoles, mitochondria, and the water-expulsion vesicle (contractile vacuole) are described. A canicular system opening into the water-expulsion vesicle contains tubules about 600 A in diameter that are lined with a filamentous material. The tubules are continuous with unlined vesicles or ampullae of larger diameter. Centrioles were not observed, but cytoplasmic microtubules radiate from a dense material similar to centriolar satellites and are frequently centered in the Golgi complex. Cytoplasmic reserve materials include both lipid and glycogen, each of which amounts to about 10% of the dry weight.     

Phagotrophy and Two New Structures in the Malaria Parasite Plasmodium berghei

Blood collected from rats infected with Plasmodium berghei was centrifuged and the pellet was fixed for 1 hour in 1 per cent buffered OsO₄ with 4.9 per cent sucrose. The material was embedded in n-butyl methacrylate and the resulting blocks sectioned for electron microscopy. The parasites were found to contain, in almost all sections, oval bodies of the same density and structure as the host cytoplasm. Continuity between these bodies and the host cytoplasm was found in a number of electron micrographs, showing that the bodies are formed by invagination of the double plasma membrane of the parasite. In this way the host cell is incorporated by phagotrophy into food vacuoles within the parasite. Hematin, the residue of hemoglobin digestion, was never observed inside the food vacuole but in small vesicles lying around it and sometimes connected with it. The vesicles are pinched off from the food vacuole proper and are the site of hemoglobin digestion. The active double limiting membrane is responsible not only for the formation of food vacuoles but also for the presence of two new structures. One is composed of two to six concentric double wavy membranes originating from the plasma membrane. Since no typical mitochondria were found in P. berghei, it is assumed that the concentric structure performs mitochondrial functions. The other structure appears as a sausage-shaped vacuole surrounded by two membranes of the same thickness, density, and spacing as the limiting membrane of the body. The cytoplasm of the parasite is rich in vesicles of endoplasmic reticulum and Palade's small particles. Its nucleus is of low density and encased in a double membrane. The host cells (reticulocytes) have mitochondria with numerous cristae mitochondriales. In many infected and intact reticulocytes ferritin was found in vacuoles, mitochondria, canaliculi, or scattered in the cytoplasm.     

Entamoeba histolytica: correlation of assessment methods to measure erythrocyte digestion, and effect of cysteine proteinases inhibitors in HM-1:IMSS and HK-9:NIH strains

Entamoeba histolytica trophozoites are able to degrade human erythrocytes; the loss of erythrocyte cellular matrix and degradation of plasma membrane were observed, along with the decrease in the average size of digestive vacuoles. Ninety-six percent of hemoglobin ingested was hydrolyzed by trophozoites within 3h, as evidenced by electrophoresis. Accordingly, X-ray spectroscopy revealed the presence of iron inside vacuoles after erythrophagocytosis, the concentration of which decreased to control levels in a similar period. Quantification of erythrocyte digestion at the early and late periods was determined by a spectrophotometric procedure, with t(1/2)=1.67 h and 35-min for HM-1:IMSS and HK-9:NIH trophozoites, respectively. In the latter, activity was due to the combined action of intracellular enzymatic activity and exocytosis. E-64c and leupeptin totally inhibited erythrocyte digestion within a 3-h period, thereafter hydrolysis took place at lower rate. Our results suggest that erythrocyte digestion in E. histolytica proceeds in different ways in these two amebic strains, and can be blocked by proteinase inhibitors.     

ASCOPHYLLUM NODOSUM (PHAEOPHYTA) IN AXENIC CULTURE AND ITS RESPONSE TO THE ENDOPHYTIC FUNGUS MYCOSPHAERELLA ASCOPHYLLI AND EPIPHYTIC BACTERIA1

Ascophyllum nodosum (L.) Le Jol. was freed from bacteria and the endophytic fungus Mycosphaerella ascophylli Cotton by repeated treatment with chlorine solutions and grown in artificial seawater. Two types of axenic culture of different origin were obtained. Type 1 was developed from apices of A. nodosum collected in the sea. Type 2 was from plants which developed from adventitious embryos on rhizoids formed by type 1. This is the first time A. nodosum has been cultivated axenically. Growth of the axenic alga was increased by IAA, 21P and zeatin. Without external growth regulators some strains of the axenic alga deteriorated within a year; others developed a filamentous habit. Sulfur in a reduced state also stimulated growth. Addition of either glucose, mannose or mannitol to the medium caused the formation of calluslike layers of loosely packed colorless cells under the epidermis of the thalli and the epidermis was sloughed off. No increase in thallus length was noticed. Mycosphaerella ascophylli in axenic culture did not excude any substances stimulating growth of the alga, but that does not exclude an influence of the fungus on the alga in vivo. The fungus, when growing within the alga, seemed to have little influence on algal morphology. A bacterized but fungus-free A. nodosum was cultivated in an artificial seawater for 8 years. In the bacteria-free alga, the fungus protruded from the epidermis and evidently utilized the alga as a carbon source. The bacteria thus seem much more important than the fungus for normal growth of the Ascophyllum plant.     

An umbraviral protein,involved in long-distance RNA movement,binds viral RNA and forms unique,protective ribonucleoprotein complexes

Umbraviruses are different from most other viruses in that they do not encode a conventional capsid protein (CP); therefore, no recognizable virus particles are formed in infected plants. Their lack of a CP is compensated for by the ORF3 protein, which fulfils functions that are provided by the CPs of other viruses, such as protection and long-distance movement of viral RNA. When the Groundnut rosette virus (GRV) ORF3 protein was expressed from Tobacco mosaic virus (TMV) in place of the TMV CP [TMV(ORF3)], in infected cells it interacted with the TMV RNA to form filamentous ribonucleoprotein (RNP) particles that had elements of helical structure but were not as uniform as classical virions. These RNP particles were observed in amorphous inclusions in the cytoplasm, where they were embedded within an electron-dense matrix material. The inclusions were detected in all types of cells and were abundant in phloem-associated cells, in particular companion cells and immature sieve elements. RNP-containing complexes similar in appearance to the inclusions were isolated from plants infected with TMV(ORF3) or with GRV itself. In vitro, the ORF3 protein formed oligomers and bound RNA in a manner consistent with its role in the formation of RNP complexes. It is suggested that the cytoplasmic RNP complexes formed by the ORF3 protein serve to protect viral RNA and may be the form in which it moves through the phloem. Thus, the RNP particles detected here represent a novel structure which may be used by umbraviruses as an alternative to classical virions.     

The mechanism of formation of inhibitor-induced ribosome helices in Entamoeba invadens

Helices andaggregates of helices (chromatoid bodies) composed of ribosomelike particles appear in cysts and slow-growing trophozoites of Entamoeba invadens. We found that similar helix aggregates were formed abundantly in actively growing E. invadens trophozoites treated with a variety of direct or indirect inhibitors of protein synthesis. The inhibitor-induced helices appeared cytochemically and ultrastructurally identical to those seen in cysts. Numerous single helices and small arrays occurred randomly distributed throughout the trophozoite cytoplasm within 15 min after treatment with NaF, which rapidly and completely stopped all nucleic acid and protein synthesis. Cycloheximide (CH), which inhibited protein synthesis as effectively a NaF, stimulated aggregate formation more slowly, and only after a delay of 30-60 min. CH temporarily blocked NaF-stimulated aggregated formation. Aggregation was slowest with actinomycin-D, which strongly inhibited RNA synthesis but depressed protein synthesis only slowly. These results suggested that release of ribosomes from mRNA was required for aggregation. Inhibition by CH was reversible, and aggregates disappeared from CH-treated amebas shortly after they were transferred to inhibitor-free frowth medium. There was no evidence that helices assembled about a structural organizer within the cell or that the process involved metabloc activity. It was concluded that the inhibitor-induced helices were composed of mature, normally functional ribosomes and that helix formation was a spontaneous and reversible consequence of the accumulation withing the cell of free monosomes (or subunits) which were prevented from binding to mRNA.     

Evaluation of membrane-bound black bodies in trophozoites and cysts of Naegleria spp

Various Naegleria strains were examined to determine the possible origin and significance of membrane-bound black bodies that were found in all exponentially growing cell populations. The bodies, 40–80 nm in diameter, were distributed randomly in the cytoplasm of Naegleria with ultrastructural features typical of trophozoites. No evidence was obtained that the contents of the black bodies were synthesized in the rough endoplasmic reticulum (ER) and packaged by membranous components, which could be a primitive “Golgi complex” in these amoebae. Examination of cells in various stages of encystment indicated that at least some of the cyst wall material was synthesized and packaged by the rough ER. After condensation into amorphous granules in the cisternae, the cyst wall material appeared in vesicles of the rough ER; these were frequently seen in close proximity to the cell membrane in the vicinity of developing cyst wall. Amorphous granules (～100 nm in diameter), which had variable densities and did not appear to be membrane bound, were seen in the cytoplasm of encysting cells. The substance of these granules also seemed to be incorporated into the cyst wall. The membrane-bound black bodies appeared to be destroyed in lysosomal elements during encystment. The membrane-bound black bodies were concluded to be characteristic of trophozoites and unrelated to encytment of Naegleria.     

Localisation of thiamine pyrophosphatase within the cytoplasmic fine structure of trophozoites of Entamoeba histolytica and E. invadens

McCaul T.F. and Bird R.G. 1978. Localisation of thiamine pyrophosphatase within the cytoplasmic fine structure of trophozoites of Entamoeba histolytica and E. invadens. International Journal for Parasitology8: 501–506. The distribution of thiamine pyrophosphatase (TPPase) activity was studied in both formaldehyde and glutaraldehyde fixed trophozoites of Entamoeba histolytica and E. invadens. The activity was localised within certain vacuoles. No dense deposits for TPPase activity were seen in the small vesicles, elongated smooth-walled lacunae equated with endoplasmic reticulum, or the nucleus. The demonstration of small vesicles surrounding the larger vacuoles indicated that the Golgi-like vacuoles might be involved in the production of cell coat materials and primary lysosomes.     

Temporal movement of digestive vacuoles in fedTetrahymena pyriformis GL-9

Summary Studies using cells ofTetrahymena pyriformis GL-9 (which had been grown in and maintained in proteose-peptone/yeast extract medium and then presented first with 0.48 m diameter latex particles for 70 minutes and then with excess carmine particles, or with the particles added in reverse sequence) have shown that there is temporal sequencing in the movement of digestive vacuoles. Under the conditions employed the digestive vacuoles are egested from the cells in approximately the order in which they were formed.