bluuebunny/crossref_metadata_2025 huggingface dataset:
Three-dimensional reconstruction of organelles inEuglena gracilisz.: I. qualitative and quantitative changes of chloroplasts and mitochondrial reticulum in synchronous photoautotrophic culture
Authors: Pellegrini Max
Published: April 2021

Ultrastructural changes of chloroplasts and mitochondria have been observed in synchronously growing cells of Euglena gracilis Z, under photoautotrophic conditions. Application of the serial section technique allows estimation of the number and volume of these organelles. Several 3-dimensional reconstructions reveal their shape and distribution throughout the cell cycle.

In young cells 10 separate diskoid or branched chloroplasts are found. They show the typical lamellar structure of euglenoid chloroplasts. During the growth phase (light period), they enlarge and their volume doubles. Some of them branch out, so that 20 lobes are formed. Thylakoids grow longer without change in number. The pyrenoid persists only during the first half of this period. During the cell division phase (dark period), branched chloroplasts divide along 2 planes which are perpendicular to each other and perpendicular to the thylakoid plane. All thylakoids are cut and their number does not change in the daughter chloroplasts. The plastidome volume constitutes 15–18% of the total cell volume over the entire life cycle.

One of the most significant observations in this report is the presence of a single permanent mitochondrial reticulum during the whole cell cycle. This giant mitochondrion consists of an extremely branched network with delicate threads (0·4–0·6 μm thick) surrounding the chloro-plasts, nucleus and reservoir. It extends throughout the cell. During the growth phase, it becomes gradually longer and doubles in volume. The degree of branching increases but the thickness of the threads remains constant. During the division phase, the mitochondrial elements appear more restricted (0 ×4 μm thick) and the reticulum becomes progressively partitioned into 2 daughter networks. At any time of the cell cycle, the chondriome volume is about 6% of the total cell volume.

These results are discussed in comparison with numerous relevant papers on fight and electron microscopy of animal and plant cells. They suggest that the descriptions of several authors on the plastidial cycle and the mitochondrial cycle in Euglena, both said to be characterized by alternate reticulate and fragmentary states, arise in part from questionable interpretation of random sections. It is evident that the form and distribution of organelles can be determined more precisely by serial sectioning.
Wall regeneration by plasmolysed cells from tissue suspension cultures of sphaerocarpos donnellii
Authors: Grusak Michael A. , Thomas Robert J. , Marsh Barry H.
Published: April 2021

De novo synthesis of wall material by cells of Sphaerocarpos donnellii was followed using radioactive tracer techniques. Uniform uptake of [14C]glucose by plasmolysed suspension cultures was demonstrated autoradiographically. Liquid scintillation counts of acid- or alkali-soluble wall carbohydrates indicate that hemicellulose and cellulose make up the major wall fractions in control and plasmolysed tissues. The amount of label in walls of plasmolysed cells was significantly lower than in those of controls. Higher relative radioactivity in pectin and hemicellulose, and lower radioactivity in cellulose were found in plasmolysed tissue. These compositional changes may enhance wall stability by providing numerous sites for cross-linkage between cellulose microfibrils of regenerated walls.
Variations in the distribution and migration of centriole duplexes in mitotic ptk2 cells studied by immunofluorescence microscopy
Authors: Aubin Jane E. , Osborn Mary , Weber Klaus
Published: April 2021

The localization and migration of centriole duplexes have been studied in PtK2 cells by indirect immunofluorescence microscopy using specific tubulin antibodies. The study demonstrated the usefulness of the immunofluorescence technique to quantitate studies of centriole migration and concomitant events such as cytoplasmic microtubule breakdown in large populations of cells. Centriole duplex locations in normal and Colcemid-treated interphase populations have been compared with duplex locations in prophase cells. A higher percentage of duplexes were found close to the nucleus in prophase than in interphase cells, but approximately 5% of the duplexes remained in the cytoplasm far removed from the nucleus in prophase and throughout the course of duplex separation. Duplex separation occurred along a wide variety of paths and duplexes did not have to be closely juxtaposed to the nuclear envelope for separation to occur. Some duplexes separated in the cytoplasm with no detectable nuclear attachment, with spindles forming far to the side of the condensing chromosomes. The timing of duplex separation did not always coincide either with chromosome condensation or with nuclear membrane breakdown, and in a small percentage of the cells separation occurred as late as prometaphase. These data suggest that normal spindle formation can occur despite the large variability in initial and final centriole duplex location, their migration patterns, and the timing of the different events. Breakdown of cytoplasmic microtubules began in prophase and progressed until prometaphase; the last cytoplasmic microtubules disappeared soon after the loss of the nuclear membrane.
The polypeptides of rat liver nuclear envelope: I. examination of nuclear pore complex polypeptides by solid-state lactoperoxidase labelling
Authors: Richardson Jonathan C. W. , Maddy Alun H.
Published: April 2021

Purified nuclei retaining a high degree of ultrastructural integrity were isolated by conventional centrifugation techniques. The cytoplasmic surface of these nuclei was iodinated using lactoperoxidase immobilized onto giant Sepharose beads; thus the outer nuclear membrane and the cytoplasmic surface of nuclear pore complexes were selectively labelled. Pore complexes in association with a fibrous lamina were isolated from these nuclei by removal of the nucleoplasm and extraction with Triton X-100. The chemical composition of the pore-lamina fraction was 93·6% protein, 6% RNA, 0.4% phospholipid. The labelling suggests that major polypeptides Ni (70000) and N2 (67000) and more than 10 other more minor polypeptides, ranging from 3 3 000 to 200 000 mol. wt, as being components of the nuclear pore complex. Polypeptide N3 (58000) is shown to be present only on the nucleoplasmic face of nuclear envelopes, probably in the fibrous lamina.
The polypeptides of rat liver nuclear envelope: II. comparison of rat liver nuclear membrane polypeptides with those of the rough endoplasmic reticulum
Authors: Richardson Jonathan C. W. , Maddy Alun H.
Published: April 2021

Nuclear envelopes are separated into pore-lamina and membrane sub-fractions by extraction in 2.0% Triton X-100 followed by pelleting of the pore-laminae. The polypeptides of these subfractions are then compared with those from isolated rough endoplasmic reticulum. The dispositions of individual polypeptides in the cytoplasmic surface of nuclear envelopes and rought endoplasmic reticulum were studied by lactoperoxidase-catalysed iodination.

These studies show that although the nuclear membranes exhibit several homologies with the Triton-soluble polypeptides of the rough endoplasmic reticulum the relative proportion of individual polypeptides within the two systems are very largely different. The cytoplasmic surfaces of the 2 membrane systems show only 2 obvious homologies at 10 5 00 0 and 15 000 mol. wt and the overall impression is that, at least in rat liver, the outer nuclear membrane is very substantially differentiated from rough endoplasmic reticulum. It is concluded that the nuclear membranes may not be regarded as a mere continuum of the endoplasmic reticulum, but should be seen as a highly specialized membrane system in their own right.
P2: A behavioural mutant of Dictyostelium discoideum
Authors: Frantz Catherine E.
Published: April 2021

Pa is a mutant strain isolated from the cellular slime mould, Dictyostelium discoideum strain NC-4. Pa differs from NC-4 in 3 major respects. Pa lacks the cyclic AMP chemotactic and signalling properties and the aggregative development of NC-4. P2 development consists of slow differentiation of resistant microcysts, which have not previously been reported in D. discoideum. Most important, Pa amoebae display a novel pattern of movement, quite distinct from that of NC-4 amoebae and any other related amoebae for which data are available.

Pa amoebae move on agar at a mean velocity of 13–3 μm/min, almost twice as fast as NC-4 amoebae. Pa amoebae have a persistence time, or directional memory ‘half-life’, of 13·3 min, over 3·5 times the NC-4 value. However, this measure is based on straight-line movement, and actual Pa persistence may be much greater because Pa amoebae move almost always in gradual left turns and left loops interspersed with occasional long, straight segments.

When Pa amoebae are plated in drops on agar, they migrate away in all directions, and the expanding drop edge can move at nearly the mean individual cell velocity. Spiral bands of amoebae can be seen in the expanding area of the drop; these invariably unwind from the original drop in a counterclockwise direction. The persistent left-turning behaviour of individual Pa amoebae is probably the major cause of this pattern of movement from drops. It also probably helps explain the similar coordinated banding behaviour observed in fields of cells. Amoebae move long distances in bands of contacting or separated cells, with no apparent organizing centre. Statistical analysis shows that Pa amoeb g have some tendency to cluster, and cell-cell contact interactions may be strong enough to promote band formation.
An ultracytochemical study of nucleolar organization in meristematic plant cells (Allium porrum)
Authors: Luck B. T. , Lafontaine J. G.
Published: April 2021

The interphase nucleolus in Allium porrum, as in many of the plant species studied so far, is highly heterogeneous in ultrastructure owing to the presence of coarse, contorted, thread-like structures, or nucleolonemata. Each nucleolonema appears to be sharply twisted and to give rise to a skein within the nucleolar mass. In order to characterize further these nucleolar components, a variety of cytochemical techniques were exploited. For that purpose, specimens were mostly fixed in 4% formaldehyde and stained in the block according to procedures known to reveal the presence of nucleic acids or proteins. Certain specimens were also digested with deoxyribonuclease, ribonuclease or proteinase K before staining. By staining with phosphotungstic acid or bismuth oxynitrate, the presence of a high concentration of proteins can be demonstrated within thin (0.15 µm), filamentous structures which are believed to correspond to the outer region of the nucleolonema. Such convoluted formations disappear upon sufficiently long extraction with proteinase K. Using Bernhard’s regressive staining technique for chromatin, the distribution of this substance throughout the nucleolar mass was found to match closely that of the nucleolonemata as revealed by several other procedures. As a last test for investigating the cytochemical make-up of the nucleolus, blocks of tissues were stained with 3,3’-diaminobenzidine, a substance known to react specifically with nucleic acids. When such specimens are digested with ribonuclease for 1 h, there persist within the nucleolus, fibrillo-granular zones the localization of which is highly reminiscent of that of the nucleolonemata. Combination of ribonuclease hydrolysis with subsequent treatment with proteinase K (30 min) induces the extraction of a large proportion of the nucleolar material, the persisting loose and rather evenly distributed fibrils exhibiting a diameter of 3-5 nm. The possibility is considered that these units may correspond to chromatin fibrils although they have most likely been displaced from their original localization during the extraction procedures.

Our cytochemical data suggest that, in Allium porrum, the nucleolonema is approximately 0.3 µm in diameter and may consist of a central axis from which chromatin loops project radially. A possible interpretation for the presence of protein-rich, 0.1 µm-thick, annular structures throughout the nucleolonemal skein is that the newly synthesized RNP products are accumulated transiently at the extremities of these loops before migrating to the immediately adjacent granular nucleolar zones.
Aggregation properties of chondrocytes infected with a temperature-sensitive mutant of rous sarcoma virus
Authors: Tanaka Akio , Kaji Akira
Published: April 2021

Aggregation capacity of chicken embryo chondrocytes decreases when transformed by Rous sarcoma viruses. Cell-to-cell aggregation capacity of chondrocytes infected with a T class temperature-sensitive mutant (tsNY68) (with the temperature-sensitive lesion at the src gene) of Rous sarcoma virus is dependent upon the temperature at which these cells are grown. When grown at the permissive temperature (36 °C), where the transforming gene is expressed, aggregation capacity was lower than normal while infected cells grown at the non-permissive temperature (41-5 °C) had similar capacity to aggregate to that of normal chondrocytes. However, after a prolonged period of culture (10 days), chondrocytes transformed by wild type SR-RSV regained the normal level of aggregation capacity. Cells transformed by tsNY68 and incubated at the permissive temperature for 10 days also regained the normal level of aggregation capacity. It appears therefore that RSV-transformed chondrocytes first become less adhesive but during long-term cultivation they regain their property to aggregate. The decrease of aggregation capacity due to T class mutants of RSV at 36 °C is dependent on constant maintenance of protein synthesis because addition of cycloheximide restored the aggregation capacity even at the permissive temperature.
The occurrence of period IV elementsin dinoflagellate chromatin: an X-ray microanalytical study
Authors: Kearns L. P. , Sigee D. C.
Published: April 2021

X-ray microanalysis of unfixed frozen-dried cryosections, and of unfixed cryosections collected by a sucrose drop technique, revealed the presence of Ca, Mn, Fe, Ni, Cu and Zn (Period IV, Periodic Table) in association with the permanently condensed chromosomes of Prorocentrum micans.

All of these metals, with the exception of Mn, routinely occurred in the chromatin of cells that had been fixed in glutaraldehyde, ethanol-dehydrated, and resin-embedded. Their detection at high levels in chemically dehydrated chromatin of Prorocentrum micans, and 3 other dinoflagellate species cultured in the laboratory, suggests they are important as bound (insoluble) components of the chromatin.

Detailed analysis of the fixed, ethanol-dehydrated material showed that for these particular cultures, each species had its own characteristic mass fraction pattern of chromatin-bound elements which differed markedly from that of the other species. Calculation of relative numbers of atoms from the mass fraction data showed that for each species about 40–50 atoms of bound Period IV elements were present per too atoms of phosphorus. This is approximately equivalent to one Period IV atom per 2 DNA nucleotides or per base pair.

Although considerable variation exists between species in terms of individual element levels, the total number of atoms of Period IV elements relative to phosphorus is remarkably constant.
Studies on the surface properties of hybrid cells: I. Sialyl-transferase activity in homogenates of malignant and non-malignant cells
Authors: Atkinson M. A. L. , Bramwell M.E.
Published: April 2021

We report here a method for the assay of the sialyl-transferase activity in crude homogenates of a wide range of cell lines growing in tissue culture. Our results indicate that particulate preparations from both malignant and non-malignant cells show a Km of 0·25 mM towards CMP-sialic acid in the presence of an excess of glycoprotein acceptor. There appear to be increased amounts of the enzyme associated with the preparations from malignant sources which are reflected in an increase in the apparent Vmax of these.

The elevated level of sialyl-transferase activity seen in the malignant cell populations is, paradoxically, associated with a decrease in the amount of bound sialic acid associated with both the whole cell homogenate preparations and the surface of these cells.
Studies on the surface properties of hybrid cells: II Sialyl-transferase activity on the surface of malignant and non-malignant cells
Authors: Atkinson M. A. L. , Bramwell M. E.
Published: April 2021

A measurable surface activity of sialyl-transferase is demonstrated by a number of different methods to exist on the plasma membranes of both malignant and non-malignant cells. The amount of enzyme present on the surface of malignant cells is found to be higher than that on the non-malignant ones in a wide range of malignant and non-malignant cell lines. It is proposed that the difference in apparent activity results in part from the presence of incomplete glycoproteins in the surface membranes of the malignant cells and in part from an increased rate of membrane synthesis in these cells.
Surface binding and uptake of polycationic ferritin by neonatal piglet intestinal epithelium
Authors: Chase Carol C. , Munn Edward A.
Published: April 2021

Pieces of small intestine from newborn, 1- and 6-day-old piglets were incubated in vitro and ligated segments were incubated in vivo with polycationic ferritin (PCF) to determine the distribution and possible role in protein uptake of anionic sites on membranes at the luminal surfaces of the epithelial cells. Most PCF binding to the absorptive cells occurred on the upper third or half of the villi and to some non-absorptive cells (tuft cells) throughout the length of the villi.

Pieces of intestine which were fixed before incubation had PCF on microvilli and apical invaginations of absorptive cells, but none in the sub-apical tubules. When samples were incubated with PCF in vitro before fixation PCF was bound to the surfaces of microvilli of absorptive cells and to the membranes lining the apical invaginations, some sub-apical vesicles and some sub-apical tubules in all age-groups. In vivo experiments with longer incubation times resulted in a similar distribution, with increased amounts of PCF in the sub-apical tubules in samples from newborn and 1-day-old piglets only. In the newborn there were small vesicles containing PCF which had apparently moved further into the cells. At the same concentration (2 mg/ml) ferritin did not enter the sub-apical tubules; but it did enter and was taken up by the cells in the presence of 4 % (w/v) serum albumin.

It is concluded that the mechanism of protein uptake does not involve the microvillar membrane and that non-specific interaction with the invaginated plasma membrane, as a function of charge-density, leads to opening of the sub-apical tubular system to protein. Movement further into the cell depends on other factors.
Numerical, morphological and topographical heterogeneity of the chondriome during the vegetative life cycle of Polytoma papillatum
Authors: Gaffal K. P. , Schneider G. J.
Published: April 2021

Numerical, morphological, and topographical changes in the mitochondrial inventory (= chondriome) during the vegetative (= asexual) life cycle of Polytoma papillatum were examined by means of the serial-sectioning technique. At the onset of interphase the chondriome consists primarily of one highly reticulated basket-shaped complex, which lines the periphery of the cell. Up to 3 additional fragments could be analysed, which, however, were much smaller and either spherical to ovoid, or elongated and poorly branched. During interphase growth both the size of the mitochondrial basket and the number of additional fragments (ca. 40) increases. During mitosis 1 (1. division cycle) the multiplication of the number of mitochondria gradually continues; the maximum (ca. 250) was counted at late telephase 1 and early cytokinesis 1, but it cannot be excluded that the sparser short-lived meta- and anaphase cells, which were unfortunately missed, contain an even higher number of mitochondrial units. Concomitant with mitosis 1, the mitochondrial basket is subdivided into several fragments; at the same time the mitochondria tend to form clusters and to occupy no longer merely peripheral but also more central regions. An irregular distribution of the chondriome results, with either local accumulations or local absence of mitochondria within definite regions of the cell. After completion of cytokinesis 1 the total number of mitochondria (ca. 80) is drastically reduced but it seems to increase again during the 2. division cycle. Irrespective of the number of preceding division cycles, the chondriome of young daughter cells enclosed in the sporangium usually resembles that of young interphase cells. Our results are discussed in relation to some of the current problems on mitochondriogenesis.
Arginase and sucrase potential in the fission yeast Schizosaccharomyces pombe
Authors: Benitez Tahia , Nurse P. , Mitchison J. M.
Published: April 2021

The induction potentials of 2 enzymes, sucrase and arginase, have been measured in asynchronous and synchronous cultures of the fission yeast Schizosaccharomyces pombe.

The effect on potential of inhibiting DNA synthesis in asynchronous cultures has been studied using 2 temperature-sensitive cdc mutants, one blocked in DNA replication and the other blocked in mitosis. The results show that despite inhibition of DNA synthesis, sucrase and arginase potential both continue to increase exponentially for at least a generation of growth after shifting the cdc mutants from the permissive to the restrictive temperature. A second method of inhibiting DNA synthesis, using deoxyadenosine, has also been tested. Cells treated with deoxyadenosine stop the increase in potential for a short period. However, experiments carried out using a cdc mutant together with deoxyadenosine show that the block to the increase in potential is due to a side effect of the inhibitor.

It appears that increase in potential is not dependent upon continued DNA replication, and that gene dosage does not control potential in the normal cell cycle. This conclusion is supported by measurements on mutants of different cell sizes. Potential is proportional to size (protein content per cell in asynchronous culture) and not to DNA content.

Although potential is not gene limited in normal cells, it does appear to be so in the abnormally large cells produced by a cdc block. If cdc mutants of different sizes are grown asynchronously, and DNA synthesis is inhibited by a shift to the restrictive temperature, potential continues to increase until the cells reach a critical DNA/protein ratio beyond which there is no increase in potential. This critical ratio is different for the 2 enzymes, but for each enzyme it is similar in all the mutants tested. When large cells (produced by a mutant block for 4 5 h) are shifted down in temperature, there are synchronous rounds of DNA synthesis and division and also step doublings in potential.

In synchronous cultures of wild type cells, both enzymes show a stepwise doubling of potential at 0 2 of a cycle after DNA replication. In synchronous cultures of cdc mutants blocked either in replication or in mitosis, the potential steps continue with the normal timing observed in wild type cells. This shows that the steps are not dependent on the events of the DNA-division cycle but are controlled by another mechanism. Attainment of a critical size might be part of this mechanism, but tests with size mutants argue against this.
On the involvement of the Golgi apparatus and GERL in processing of secretion products and some enzyme activities in the rat pituitary mammotroph
Authors: Nevorotin A. J.
Published: April 2021

Whole pituitary glands of Wistar albino rats were either (a) immediately fixed in an aldehyde fixative (Kamovsky), or incubated prior to the fixation in (b) unaltered medium 199, (c) medium 199 supplemented with 1·65 M sucrose, or (d) medium 199 diluted with distilled water (1 : 2). Incubation time was for 0·5 h, at a temperature of 4° or 37 °C. All specimens were routinely processed for acid phosphatase (AcPase) or thiamine pyrophosphatase (TPPase) demonstration and examined ultrastructurally. In the control series a and b AcPase activity in the mammotroph was associated with the cistemae of GERL, dense bodies, coated vesicles, immature secretion granules often continuous with the cistemae of GERL, and occasionally with the innermost lamellae of the Golgi apparatus (GA). TPPase activity was found within the innermost lamellae and the fenestrated element of GA. Occasional granular cores were seen within TPPase-positive lamellae. In all mammotrophs of series c (4° and 37 °C) severe shrinkage of the lamellae of GA and other membrane-bound structures in the Golgi area was apparent. In some mammotrophs of the series c (37 °C) AcPase activity, in addition to its usual sites, was revealed within all lamellae of GA, as well as in some tubular and fenestrated elements continuous with GA. The innermost AcPase-positive lamellae of GA were sometimes partially separated from the others and continuous with immature secretion granules or dense bodies, thus qualifying as cistemae of GERL in the course of their gradual formation from the Golgi lamellae. A gradual increase in AcPase activity from the cis to trans poles of GA could sometimes by noted. TPPase activity in this series spread over all lamellae of GA and its fenestrated elements. The occasional profiles of rough endoplasmic reticulum and some smooth-surfaced vesicles near GA also gave positive reactions for the enzyme. At 4 °C the distributions of both enzyme activities were as in the controls. In all series d (4 or 37 °C) the membrane-bounded organelles of the mammotroph were greatly swollen except for occasional cells (at 37 °C) in which all lamellae of GA were paradoxically shrunk as in series c. The distributions of AcPase and TPPase activities in series d were as in the controls, except for some cells with shrunken lamellae (at 37 °C) in which TPPase activity was spread throughout GA. The results suggest that experimental dehydration of GA in the mammotroph acts by overcoming latency of AcPase and TPPase or by accelerating maturation of AcPase and TPPase activities from their hypothetical precursors thereby tracing the intracellular pathways of these enzymes. It also follows that GA may participate in the initial stage of secretion-granule formation and enzyme-activity maturation whereas GERL finalizes the process.
The secretion of the eggshell of Schistocerca gregaria: ultrastructure of the follicle cells during the termination of vitellogenesis and eggshell secretion
Authors: Kimber Susan J.
Published: April 2021

The secretion of the eggshell by the follicle cells in the desert locust, Schistocerca gregaria, was studied using the electron microscope. The 3 layers of the eggshell, the vitelline membrane, the endochorion, and the exochorion, are produced in sequence over a short period of about 30—36 h. The follicle cells contain little rough endoplasmic reticulum (RER) and small inconspicuous Golgi bodies during vitellogenesis. As eggshell secretion approaches there is an increase in the amount of RER and number of Golgi bodies. During vitelline membrane and endochorion secretion the RER and Golgi cistemae contain electron-dense product. At each stage of the 3-phase secretion cycle the follicle cells contain Golgi bodies and secretion vesicles with distinct morphology. The follicle cells increase in breadth and decrease in height between the beginning and end of eggshell secretion. The endochorion ridges arise at the junction between follicle cells and appear to be moulded by the microvilli formed at this position. In the ovary prior to ovulation, the eggshell consists of a thin (0·5 μm) electron-dense vitelline membrane, a 5–10-μm-thick endochorion consisting of crystalline tubules in a granular matrix, and an outer fibrillar exochorion layer, 20–30 μm thick. Further changes take place in the vitelline membrane and the endochorion after oviposition, and a layer of curly fibres, the extrachorion, is secreted in the oviduct.
Chloroplast division in spinach leaves examined by scanning electron microscopy and freeze-etching
Authors: Chaly N. , Possingham J. V. , Thomson W. W.
Published: April 2021

Spinach leaf disks were cultured for 5 days in low-intensity green light and then were transferred to high-intensity white light. Harvests over the next 16 h established that cell area increased by about 80% and chloroplast number per cell increased by about 65 %, while the percentage of dumbbell-shaped chloroplasts per cell decreased by 65 %. Freeze-etch replicas of fixed and unfixed leaf disks, as well as scanning electron-microscope preparations of fixed material, contained dumbbell-shaped chloroplasts constricted to various degrees. Freeze-etch replicas of unfixed cells from young leaf bases, in which the number of chloroplasts per cell is known to be rapidly increasing, also contained many constricted chloroplasts. It is concluded that dumbbell-shaped chloroplasts occur in vivo and represent a stage in the division of chloroplasts.
Isolation of the cytoskeleton from Giardia. tubulin and a low-molecular-weight protein associated with microribbon structures
Authors: Holberton D. V. , Ward A. P.
Published: April 2021

The sucking disk of Giardia is supported by a large, plate-like organelle: the ventral disk cytoskeleton. Extraction by Triton-X 100 of Giardia trophozoites from the mouse gut, or of G. duodenalis or G. lamblia grown from cultures, yields cell-free disk cytoskeletons. Up to 8 flagellar axonemes may be attached to an isolated disk.

Disks are seen in the electron microscope to be composed of concentrically coiled microtubules bonded to microribbons. Microribbons are large, laminated structures, linked by dense networks of crossbridges. They are made up of regularly arranged subunits. Microtubules and microribbons are preserved in Triton for long periods, but crossbridges are slowly dissolved.

Whereas the addition of ATP causes axonemes to resume bending, active movements were not detected in disks. It seems more likely that a disk is a passive effector, which may be acted upon by other contractile structures of the cytoplasm. It is a highly specialized and quintessential cytoskeleton.

Disks and axonemes will dissolve in sodium dodecyl sulphate (SDS), and after SDS-gel electrophoresis 2 prominent bands are apparent. One, corresponding to tubulin, migrates in low ionic strength, high pH buffers as 2 closely spaced bands of equal staining density. The other, a smaller protein, is a complex polypeptide band of molecular weight 30000 Daltons. Allowing for staining differences, the 2 proteins are probably present in cytoskeletons in roughly equal amounts. Because of their size, microribbons account for at least 75 % of the structured material stained by electron stains in pellets of cytoskeletons. Disk and axoneme microtubules comprise the minority fraction. This result suggests that, like microtubules, microribbons are a source of structural tubulin and probably also contain the 30000 mol. wt protein.
The appearance of carbohydrate-rich material in the developing golgi apparatus of amoebae
Authors: Flickinger Charles J.
Published: April 2021

The silver proteinate reaction was used to stain carbohydrate-rich substances in normal Amoeba proteus and in the developing Golgi apparatus of renucleated amoebae. Normal cells contained stained material, which probably is glycoprotein, in the cell surface, cisternae at the concave pole of the Golgi apparatus, and cytoplasmic vesicles and vacuoles. Previous radioautographic studies had shown that glycosylation occurs in the Golgi apparatus, and that material in the Golgi apparatus is precursor to the cell surface. Amoebae were enucleated for 5 d, which results in a decline of the Golgi apparatus, the disappearance of the glycoproteincontaining cisternae preceding that of the rest of the organelle. A new nucleus was then transplanted into the enucleate amoebae, bringing about the regeneration of the Golgi apparatus. Small curved cisternae that appeared 30 min after renucleation lacked staining with silver proteinate. By i h after renucleation, however, the content of cisternae toward the concave poles of Golgi bodies stained with silver proteinate. The Golgi apparatus in cells fixed 6 h and 1 d after operation resembled that of normal amoebae in both morphology and staining pattern. The results suggest that the developing Golgi apparatus acquired the capacity to participate in assembly of cell-surface material within 1 h after renucleation. This occurred before development of the normal enzymic activity of the Golgi apparatus was completed.
Inorganic phosphate accumulation and phosphatase activity in the nucleus of maize embryo root cells
Authors: Deltour R. , Fransolet S. , Loppes R.
Published: April 2021

The nucleus of growing root cells of Zea mays contains a high concentration of inorganic phosphate. In order to verify whether this high nuclear Pt concentration is correlated with the metabolic activity of the nucleus, the P1 has been visualized in root cells of maize embryos at the electron-microscope level during 2 different periods which are both characterized by a spectacular reactivation of the nuclear metabolism, i.e. the early germination and the period of recovery following a thermal treatment given to the seeds after 48 h of germination. In both situations the P1 concentration increased in the nucleus during its reactivation.

To verify whether the high nuclear P1 concentration could be of endogenous origin, the phosphatase activities were measured in crude extracts of root tissues during nuclear reactivation. The specific activity was optimal at pH 4·5 and was shown to increase with cellular reactivation.

The ultrastructural localization of acid phosphatase activity showed that P1 may be produced at 3 distinct sites: plasmalemma, vacuoles and most probably nucleus itsef. High acid phosphatase activities were found in nuclei displaying a high metabolism.

Taking these results and previous data into account, we suggest that a correlation may exist between the rate of nuclear transcription, the level of nuclear acid phosphatase activity and the nuclear P1 accumulation.
Biophysical and morphological correlates of kinetic change and death in a starved human melanoma cell line
Authors: Sheridan J. W. , Bishop C. J. , Simmons R. J.
Published: April 2021

An investigation was made of the sequential biophysical and morphological changes that occur as cultured human melanoma cells (MM96) outgrow their supply of nutrient. Simultaneous buoyant-density and velocity-sedimentation fractionation experiments were used to characterize cells from 3 kinetically differing types of culture. Cells from exponential cultures were large, moderately dense and rapidly sedimenting; cells from post-exponential cultures were of intermediate size, less dense and much more slowly sedimenting; and dye-excluding cells from reproductively non-viable late post-exponential cultures were small, of widely variable though generally high density and sedimented moderately rapidly. Although reproductive viability was high in cells from both exponential and post-exponential cultures, depletion of clonogenic cells was seen at the extremes of the distribution profiles of cells fractionated by either method. This was particularly evident at the low-density extreme of the buoyant-density profiles where cells retained viability despite their loss of proliferative potential.

As cells became post-exponential, nuclear size diminished in parallel with cell size, the number of microvilli declined, mitochondria condensed, cytoplasm vacuolated, the frequency of osmiophilic vacuolar inclusions rose, chromatin clumped and nucleoli became prominent. Progression to a reproductively non-viable late post-exponential state resulted in a continued parallel fall in nuclear size, increased cytoplasmic blebbing, further mitochondrial condensation, an increased proportion of cytoplasmic vacuoles containing osmiophilic material, the major part of which was melanin, and further clumping and margination of chromatin. Cells progressed rapidly from this newly described pre-apoptotic state to death by apoptosis, a process characterized by the budding and division of cells into a number of ultrastructurally well-preserved membrane-bound fragments.
The movement of cell clusters in vitro. morphology and directionality
Authors: Kolega John
Published: April 2021

The movement of cells in small groups, or clusters, was studied in vitro using epithelioid cells from Gordon-Kosswig melanomas (from poecelid fish) and time-lapse cinemicrography. Tumour expiants cultured on glass yield cell sheets from which groups of cells separate and become independently motile clusters. These clusters typically contain 3–30 cells, but may have as many as 50. They propel themselves at speeds of 0·2–4·0 μm/min by means of broad hyaline lamellae. The distribution of lamellae around the perimeter of each cluster correlates with both direction and speed of cluster movement, i.e. a cluster moves with its most lamellar region at its leading edge, and the greater the extent of the leading lamellar region the greater the speed. Also, a cluster tends to keep moving in the same direction. This persistence is due to a relatively constant distribution of lamellae. Cells on the trailing edge usually lack lamellae and most are very elongate and oriented perpendicular to the direction of cluster movement. In general, whenever a cell elongates, there is a loss of lamellar activity along its taut edges, parallel to the axis of elongation. Thus, any region with less lamellar activity would tend to be elongated by the outward pull of the more active regions to either side and would, in consequence, suffer a further reduction in lamellar activity. In this way, the distribution of regions of lamellar activity is self-reinforcing and the result is persistence of movement in a particular direction. This phenomenon could play an important role in giving directionality to certain morphogenetic movements, such as neural crest cell migration.
Non-selective junctional communication between some different mammalian cell types in primary culture
Authors: Hunter Graeme K. , Pitts John D.
Published: April 2021

Intercellular transfer of tritium-labelled uridine nucleotides has been used to detect junctional communication between various cell types in primary culture. Epidermal kératinocytes, melanocytes and dermal fibroblasts from new-born-mouse skin, and epithelial cells from baby mouse kidney form communicating junctions in all possible homologous and heterologous combinations. This lack of detectable communication specificity between cells in primary culture contrasts with the specificity shown by some established cell lines.
Desmosome frequency: Experimental alteration may correlate with differential cell adhesion
Authors: Wiseman Lawrence L. , Strickler John
Published: April 2021

Differential cell adhesion, a suggested guiding force for tissue rearrangements during embryogenesis, could be affected by desmosome frequency. A model system for studying embryonic tissue-positioning behaviour involves combining different tissues and following their rearrangements. We have previously shown that for one tissue, embryonic chick heart ventricle, direction of tissue positioning can be altered experimentally. Heart tissue precultured for 2 5 days tends to segregate internally, while tissue pre-cultured for just half a day tends to segregate externally. Also, intact fragments of tissue tend to segregate internally, while reaggregates of trypsin-disaggregated tissues tend to segregate externally. We show here that treatments that increase the tendency to internalize also increase the frequency of adherens junctions and treatments that increase the tendency to externalize decrease the frequency of junctions. An identical hierarchical ordering of the 4 experimental tissues occurs with respect to positioning behaviour and desmosome frequency. In the hierarchy, 2 ·5-day-cultured fragments > 2·5-day-cultured reaggregates > 0·5-day-cultured fragments > 0·5-day-cultured reaggregates, tissues to the left tend to segregate internally and to have more desmosomes. Tissues to the right segregate externally and have fewer desmosomes. This is what is expected if desmosomes are organelles for adhesion and if differential adhesion is a factor in tissue-positioning behaviour.
Partial reversion of the transformed phenotype of murine sarcoma virus-transformed cells in the presence of interferon: A possible mechanism for the anti-tumour effect of interferon
Authors: Hicks N. J. , Morris A. G. , Burke D. C.
Published: April 2021

The effect of interferon (IFN) on a series of cellular properties which characterize the transformed state was studied, using normal mouse fibroblasts (both established lines and embryo cells) and cloned derivatives transformed by murine sarcoma virus. It was found that the transformed cells behaved in a more normal fashion in the presence of IFN. This was indicated by reduction in saturation density, decreased DNA synthesis in crowded cultures, decreased formation of foci on monolayers of normal cells, and decreased growth in soft agar. These effects were not due to generalized inhibition of cell growth, since IFN had little effect on the growth and cloning efficiency of the cells used. In the presence of IFN, cellular morphology also appeared more normal. These results suggest that growth control of transformed cells may be at least partly restored by IFN, an effect which could in part account for the antitumour effect of IFN.
The interpretation of interferencereflection images of spread cells: Significant contributions from thin peripheral cytoplasm
Authors: Gingell David
Published: April 2021

In interference-reflection microscopy, used for investigating cell-substratum separation, it is commonly believed that cytoplasmic thickness can be ignored, provided a high illuminating numerical aperture (INA) is used. It is shown here that even when a maximal INA is used, cytoplasmic lamellae of 1 μm or less can be major determinants of the image. The leading lamella of spreading tissue cells and large peripheral areas of Dictyostelium discoideum amoebae on adhesive substrata are less than 1 μm thick and it is argued that hitherto unexplained features of the interference images of these cells may be interpreted in terms of the theory used here.
Determination of secretory vesicle production rates by dictyosomes in pollen tubes of Tradescantia using cytochalasin d
Authors: Picton Jill M. , Steer Martin W.
Published: April 2021

Pollen tubes of Tradescantia were grown in vitro and exposed to 0·3 μg/ml cytochalasin D for 5 or 10 min. Fine-structural observations revealed no visible effect of the drug on the organelles. Stereological analysis, using a method recently developed by Rose (1980) to obtain sphere size-distributions corrected for section thickness, revealed a substantial increase in the number of secretory vesicles present in the cytoplasm around the dictyosomes. Equating the rate of vesicle accumulation with the rate of vesicle production, a total of 5388 vesicles per minute are formed by a growing tube. This corresponds to 2·4 vesicles per minute per dictyosome, and a turnover rate of 3·7 min for a single dictyosome cisterna, or about 15–18·5 min for a complete dictyosome. The calculated vesicle production rate agrees well with that required to sustain the observed growth rate of such tubes, based on the addition of membrane or wall material to the tube tip.
The effects of fibronectin on the adhesion and migration of chinese hamster ovary cells on collagen substrata
Authors: Schor Seth L. , Schor Ana M. , Bazill George W.
Published: April 2021

Data are presented indicating that the adhesion of Chinese hamster ovary cells (CHO) to films of denatured type I collagen occurs by a fibronectin-dependent mechanism, whereas the adhesion of these cells to 3-dimensional gels of native type I collagen fibres may occur by either a rapid, fibronectin-dependent mechanism or by a slower, fibronectin-independent mechanism. Data are also presented indicating that fibronectin promotes the migration of CHO cells on native type I collagen fibres.
The effect of various cooling rates on the membrane ultrastructure of frozen human erythrocytes and its relation to the extent of haemolysis after thawing
Authors: Fujikawa Seizo
Published: April 2021

Human erythrocytes suspended in buffered isotonic saline were frozen to the temperature of liquid nitrogen at various cooling rates of 3, 140,700, 1800,3500, 8000 and 11 500 deg. C/min. The membrane ultrastructure in the frozen state and the extent of haemolysis after thawing were examined at each cooling rate. As the cooling rates increased from 3 to 3500 deg. C/min, the extent of lysis gradually decreased, but further increase in cooling rates in excess of 8000 deg. C/min resulted in an abrupt increase of lysis. Membrane-associated vesicles devoid of intramembrane particles (IMPs) were formed in the erythrocyte membranes frozen at cooling rates slower than 1800 deg. C/min. The frequency and size of these vesicles were highly cooling-rate-dependent and they were no longer formed in the erythrocyte membranes frozen at cooling rates faster than 3500 deg. C/min. Another membrane ultrastructural change associated closely with the formation of intracellular ice crystals appeared at cooling rates faster than 8000 deg. C/min. The membrane regions in direct contact with intracellular ice crystals were physically damaged and had an appearance resembling worm-eaten spots. The erythrocytes frozen at a cooling rate of 3500 deg. C/min exhibited ultrastructural integrity of the membrane by avoiding the membrane changes caused by either slow or fast freezing. It is suggested, from the close relation between membrane ultrastructure and the extent of haemolysis, that the ultrastructural integrity of membrane in the frozen state is important for avoiding haemolysis after thawing, and that the membrane ultrastructural changes caused by both slow and fast freezing were responsible for the lysis after thawing.
Ultrastructure of sheep erythrocyte plasma membranes and cytoskeletons bound to solid supports
Authors: Lang Robert D. A. , Nermut Milan V. , Williams Lynn D.
Published: April 2021

Sheep erythrocyte plasma membrane monolayers were formed on positively charged supports by means of controlled lysis and squirting of cells so that the original protoplasmic or inner surfaces (PS) were exposed. The appearance of the surface was studied by transmission electron microscopy of platinum/carbon replicas following freeze-drying of the membranes. After gentle washing with water or dilute buffer, a network of filaments and particles was found to cover the surface.

Whole cells bound to positively charged supports were treated with Triton X-100 and hypertonic KC1, which left the protein components of the cytoskeleton on the support. Stereo electron microscopy of Pt/C replicas of these residues showed complex networks of filaments, similar to those seen on the cytoplasmic surfaces of the intact membranes. In both cases the lengths of the filaments correspond to that of the spectrin dimer.

Removal of spectrin, actin and other proteins by alkaline treatment led to loss of this network and revealed 15·4-nm particles on the membrane PS. These particles, which were also visible after negative staining, could be removed by treatment with trypsin and were found to correspond to band 4 protein (the equivalent of human erythrocyte band 3 protein). Membranes freeze-fractured following treatment with alkali showed normal intramembranous particles with frequencies similar to those of native membranes. This indicates that band 4 protein spans the sheep erythrocyte membrane and forms a very high proportion of the intramembranous particles. The protoplasmic portions of these particles may be membrane binding sites for the cytoskeleton in sheep erythrocytes.