bluuebunny/crossref_metadata_2025 huggingface dataset:
Inversion in Volvox Tertius: The effects of con a
Authors: Ireland Grenham W. , Hawkins Shirley E.
Published: April 2021

During the development of Volvox tertius spheroids, a single-celled gonidium enlarges and undergoes multiple incomplete cleavages to give an embryo which is ‘inside-out’ with respect to the adult organism. A morphogenetic movement, termed ‘inversion turns this hollow ball of cells ‘inside-out’ through a hole, the phialopore. In V. tertius this phialopore possesses 4 inwardly directed lips.

Normal inversion was studied in vitro in slide chambers and involved cell-shape changes accompanied by the production of pseudopodia and the bending backwards of the phialopore lips.

100 µg/ml Con A specifically and reversibly blocked inversion. Despite the inhibitory effect on cell division, the blocking of inversion was not due to the blocking of the last cell division some 50–100 min prior to inversion. Neither did the first cell-shape change from pear-to spindle-shape appear blocked. A feature of inhibition by Con A was the enhanced production of pseudopodia by embryos blocked at inversion, and the abnormal production of pseudopodia by embryos blocked at earlier stages. Non-inverting embryos showed internal flagella. We suggest that the Con A block to inversion, which may be reversed by α-methyl mannoside, arises from the prevention of backwards-bending of the phialopore lips.

Fluorescein-isothiocyanate-Con A bound to embryo and cell coat, and more strongly to the embryo at pre-inversion. SDS-polyacrylamide gel analysis of proteins isolated from embryos showed 4 glycoprotein bands, but Con A binding to these bands could not be demonstrated.
Differentiation of neurosensory cells in Hydra
Authors: Davis Lowell E.
Published: April 2021

The differentiation of neurosensory cells in Hydra has been studied at the level of the electron microscope. These cells arise from interstitial cells (undifferentiated cells) and not from pre-existing nerve cells. Furthermore, there is no evidence to suggest that neurosensory cells represent a stage in the development of other nerve cells, i.e. ganglionic and neurosecretory cells. Major cytoplasmic changes in fine structure during differentiation include development of a cilium and associated structures (basal body, basal plate, rootlets), development of micro tubules and at least two neurites, increase in Golgi lamellae and formation of dense droplets typical of neurosecretory droplets, structural variations in mitochondria and a decrease in the number of ribosomes. Granular endoplasmic reticulum is characteristically poorly developed in all stages of differentiation, including the mature neurosensory cell. Nuclear and nucleolar changes also occur during differentiation but these are less dramatic than the cytoplasmic events.

The possibility of neurosensory cells being bi- or multiciliated and the presence of inter cellular bridges between these cells are considered. The function of neurosensory cells is dis cussed briefly in relation to the function of the cilium and neurosecretory droplets.
Locomotion ofXenopusepidermis cells in primary culture
Authors: Bereiter-Hahn Jürgen , Strohmeier Renate , Kunzenbacher Inge , Beck Konrad , Vüth Monika
Published: April 2021

The locomotion of single epidermis cells, grown out from Xenopus laevis tadpole tails has been investigated by time-lapse cinemicrography using phase-contrast and reflection-contrast optics. The cells develop a large, mostly 200–250 nm thick, lamella, which adheres homogeneously to the supporting coverglass and exceeds the projection area of the cell body. From the comparison of RIC-pictures taken at high (1.06) and low (0.62) numerical aperture of illumination (I.N.A.) we deduce that at low I.N.A. the embossment of the medium-facing side of the lamella is visualized. By this method microcolliculi are demonstrated, which form at the edge of the lamellipodium and move backward. They resemble ruffles, but are flatter and no membrane flow towards the perinuclear region is observed. Indirect immunofluorescence reveals an enhanced staining for actin and α-actinin in the lamellipodium and in the transition region of cell body and lamella. Tonofilaments do not participate in lamella formation, the relatively few microtubules seem to be oriented in the direction of cytoplasmic flow. Electron micrographs demonstrate the course of fibrils in the cell body and a meshwork of actin filaments and membranous tubules in the lamella. Based on these findings a model for cell locomotion is presented : the motive force is generated by the cell body causing a flow of cytoplasm towards the periphery and extension of the lamella at its edge. The activity of the lamellipodium has to ensure the flat form of the advanced edge; microcolliculi are assumed to represent a small membrane store for the extension of the lamella. The lamellipodium is not involved in the production of motive force. The cell body is anchored to the lamella by radiating fibrils and the fibrillar meshwork is inserted at the ‘dorsal’ membrane of the lamella and the basal filament cortex of the cell body. This anchorage provides the structural basis for the uptake of lamella material into the cell body in the transition region.
Effects of organic solvents on flagellar asymmetry and quiescence in sea urchin sperm
Authors: Gibbons Barbara H.
Published: April 2021

Low concentrations of methanol, 2-propanol and ethylene glycol increase the asymmetry of the flagellar waveforms and the turning rate of both live sperm and potentially symmetrical sperm reactivated with 1 mM-MgATP2−, while at the same time causing a decrease in the beat frequency. Similar effects are observed if the solvents are added to preparations of potentially symmetrical sperm reactivated in the presence of 1 mM free Ca2+, or to potentially asymmetrical sperm reactivated without added Ca2+. A second group of solvents, N,N- dimethylformamide, formamide and p-dioxane, also decrease the flagellar beat frequency, but have the opposite effect on symmetry, reducing the asymmetry of the waveforms and the turning rate of potentially symmetrical sperm reactivated in the presence of 1 mM free Ca2+. These effects of solvents are all reversible within about 5 min after initial exposure to solvent.

Higher concentrations of methanol and 2-propanol (above ∼5 and 0·8 mole %, respectively) induce quiescence in potentially asymmetrical sperm reactivated with concentrations of MgATP2− ranging from 10 μM to 1 mM. The quiescent flagella initially assume a bent form very similar to that seen in Ca2+-induced quiescence, and show a subsequent time-dependent distortion of the initial bent form with eventual disintegration and splitting off of bundles of microtubules.

Dimethylformamide, formamide and dioxane have almost no effect on the intrinsic asymmetry of potentially asymmetrical sperm reactivated in the absence of added Ca2+,butaddition of these solvents to potentially asymmetrical sperm that have been induced to become quiescent by addition of 0·1 mM free Ca2+ causes the sperm to resume swimming with flagellar waveforms that are substantially more symmetrical than those of the starting preparation before the addition of Ca2+.

Mild digestion with trypsin of reactivated sperm that have been induced either to beat asymmetrically or to become quiescent by addition of methanol causes a gradual appearance of symmetrical flagellar beating, as in the case of Ca2+-induced quiescence. The flagellar beat frequency, however, remains low, at about 20 Hz.

The results suggest that the solvents either mimic or block the action of Ca2+ by interaction with a Ca,+-dependent regulatory protein, and may also induce alteration in the rate constants of dynein ATPase.
Age-associated change in macronuclear DNA content in Paramecium Caudatum*
Authors: Takagi Yoshiomi , Kanazawa Naoko
Published: April 2021

Macronuclear DNA content in Paramecium caudatum was found to be almost unchanged with a mean of about 400C during the earlier two-thirds of the life span in terms of the number of fissions and then it dropped rapidly to about one-fifth of the initial content. The age when rapid DNA decline occurred corresponded to that when the characteristics of senescence appeared. This decreasing pattern of macronuclear DNA content contrasted with earlier observations in P. tetraurelia, P. bursaria and Tetrahymena thermophila. The data suggested that in P. caudatum the distribution pattern of macronuclear DNA to daughter cells changed from an almost equal distribution in younger cells to an unequal distribution in older cells, while the relative volume of the macronucleus to the whole cell remained almost constant throughout the life cycle.
Interaction between intracellular vacuoles and the cell surface analysed by finite aperture theory interference reflection microscopy
Authors: Gingell David , Todd Ian , Owens Norman
Published: April 2021

Using finite aperture theory we have shown that localized very dark areas in the interference reflection images of Dictyostelium discoideum amoebae are due to the close intracellular approach of vesicles and tubular elements of the contractile vacuole system to the plasma membrane adjacent to the substratum. Vesicles interacting in this way become locally deformed to the planar contour of the substratum and are separated from the cell surface membrane by a constant ≲ 0·1 μm of cytoplasm. Lamellar processes formed by these cells on very adhesive surfaces have identical dimensions. This minimal thickness may be a mechanical consequence of a contractile mechanism which pulls membranes together.
Cell shape and organization of cytoskeleton and surface fibronectin in non-tumorigenic and tumorigenic rat liver cultures
Authors: Bannikov G. A. , Guelstein V. I. , Montesano R. , Tint I. S. , Tomatis L. , Troyanovsky S. M. , Vasiliev J. M.
Published: April 2021

Morphological changes associated with neoplastic transformation of epithelial cells were studied in a series of IAR cell lines derived from rat liver. The series included three independently obtained, non-tumorigenic lines and five derived, tumorigenic lines.

The morphology of cell surfaces was observed by scanning electron microscopy; the distribution of actin, tubulin and fibronectin was determined by indirect immunofluorescence.

All the non-tumorigenic lines had a typical epithelioid morphology: isolated cells of these lines spread on the substratum had a discoid shape and contained circular, marginal bundles of microfilaments and microtubules. In denser areas, the cells formed monolayered sheets with characteristic marginal bundles of microfilaments near the free edges.

Decreased spreading of isolated cells on the substratum was the characteristic feature that distinguished tumorigenic lines from their non-tumorigenic parent lines. In particular a decrease in the size of the ring-like, peripheral lamella and its disintegration into several discrete lamellar zones were often observed; as a result, the cell shape was altered from discoid to polygonal or elongated. The altered distribution of microfilament bundles and microtubules was characteristic in elongated cells; the pattern of the cytoskeletal elements of these cells resembled that of polarized fibroblasts. Complete disappearance of microfilament bundles was observed in cells of only one tumorigenic line.

Various degrees of disorganization of monolayered cell sheets were observed in tumorigenic cultures, accompanied by an altered distribution of microfilament bundles.

The alterations in the fibronectin-containing structures were more complex: there were often fewer fibronectin ‘spots’ and fibrils at the lower surfaces of cells of tumorigenic cultures as compared with those of non-tumorigenic ones; there were more fibrils in dense cultures of certain lines but fewer in others.

It is concluded that alterations in the ability to spread on the substratum and to form cell–cell contacts are common features of morphologically transformed fibroblastic and epithelial cultures. However, the actual changes in the cytoskeletal structures that accompany these alterations are different in transformed cultures of various tissue types.
The Euglena paraflagellar rod: structure, relationship to other flagellar components and preliminary biochemical characterization
Authors: Hyams Jeremy S.
Published: April 2021

Demembranated flagella from Euglena gracilis consisted of three distinct components: a 9 + 2 axoneme of microtubules, an extensive surface coating of long and short mastigonemes and a lattice-like axial fibre known as the paraflagellar rod. Negatively stained preparations of isolated paraflagellar rods showed the major structural unit to be a 22 nm filament oriented at approximately 45° to the long axis of the rod in a 7-start left-handed helical arrangement. Attachment of the paraflagellar rod to the flagellar axoneme was via a series of goblet-shaped projections, which anchored the rod to a single outer doublet microtubule. Comparisons of axonemes from E. gracilis bearing the paraflagellar rod with those of Chlamydomonas rein-hardtii, in which it is absent, by polyacrylamide gel electrophoresis revealed the presence of two prominent additional peptides with molecular weights of 80000 and 69000. Exposure of Euglena axonemes to trypsin selectively solubilized the paraflagellar rod and also removed both proteins, which were therefore tentatively identified as the major subunit proteins of the rod and designated PFR 1 and 2, respectively. In addition to these details of the structure and composition of the paraflagellar rod, the mode of attachment to the axoneme of both long and short mastigonemes was also identified.
The relationship between chromosome volume and dna content in unsquashed metaphase cells of barley, Hordeum vulgare cv. Tuleen 346
Authors: Bennett M. D. , Smith J. B. , Ward J. P. , Finch R. A.
Published: April 2021

The present work used haploid and diploid cells of barley, Hordeum vulgare L. cv. Tuleen 346 (2n 2x =14), which has three reciprocal translocations. All seven chromosomes of the haploid set are distinguishable using morphological criteria in Feulgen-stained root-tip squashes seen in the light microscope, as are five of the bivalents at diakinesis. The relative DNA content per bivalent was estimated in pollen mother cells at diakinesis. The results showed that all seven chromosomes or bivalents of Tuleen 346 can be identified using relative DNA content as sole criterion. The absolute and relative volumes of the seven chromosomes were estimated from electron micrographs of serial sections of unsquashed root-tip cells of a haploid. The results show that, using relative chromosome volume as sole criterion, it is highly probable that all seven chromosomes in single unsquashed cells of Tuleen 346 can be correctly identified. Consequently, tests for various non-random spatial arrangements of chromosomes in unsquashed cells of Tuleen 346 using this character to identify the chromosomes should be feasible. There was a very highly significant positive relationship (r>0·99) between relative chromosome volume and mean relative DNA content per chromosome for each cell examined at metaphase of mitosis or meiosis. Thus, some mechanism ensures that the degree of condensation of all seven chromosomes within a cell is usually very similar in Tuleen 346, lespite its grossly abnormal karyotype.
Cell wall texture along the growth gradient of the mung bean hypocotyl: Ordered assembly and dissipative processes
Authors: Roland Jean-Claude , Reis Daniele , Mosiniak Michele , Vian Brigitte
Published: April 2021

A step-by-step analysis of wall characteristics was performed along a defined growth gradient in order to follow the dynamics of changes in wall organization according to the state of elongation of the cell. Specimens were taken from the elongating zone of 3-day-old mung bean (Vigna radiata L. Wilczek) hypocotyls grown in constant culture ∞nditions. A technical procedure was developed in order to perform a complete series of observations along the growth gradient of individual hypocotyls and at the same time to locate the levels in relation with the growth state. Weight and thickness measurements were performed in parallel with observations of changes in wall texture using ultrastructural cytochemistry (periodic acidμhiocarbohydrazide/silver proteinate test for polysaccharides associated with dimethylsulphoxide and methylamine extractions). Ultrastructural observations were made on cortical cells, mainly on epidermal cells.

For a cell that runs through the complete growth process the preliminary phase is a massive elaboration of wall subunits. The wall thus built is highly ordered (twisted plywood). Both thickness and order are maximal when the cell reaches the exponential phase of extension. An unstable balance exists between ordered assembly (with new layer deposition) at the inner wall, and dispersion of subunits (resulting from extension) at the outer wall. Growth takes place at the expense of thickness and the pre-established order. The balance is rapidly shifted and the dispersion of the subunits progressively predominates. Wall construction being no longer continued, elongation slows down and stops.

The wall acts as a dissipative structure changing from order to a subsequent irreversible disorder. In terms of growth capacity, the more ordered the wall is, the more extendible is the cell. The cessation of growth occurs when order has been completely lost. The results indicate that apparently conflicting concepts proposed in the literature, for the organization of the wall during cell elongation, are not mutually exclusive in the tissues studied.
Subpellicular microtubules of Euplotes eurystomus: their geometry relative to cell form, surface contours and ciliary organelles
Authors: Grim J. Norman
Published: April 2021

There is a layer of microtubules (MT) beneath the innermost pellicular membrane of the ciliated protozoan Euplotes eurystomus. These MT have been revealed for scanning electron microscopic study by chemical dissection techniques. In much of the body of this ciliate, these MT are oriented parallel to its long axis. Those directed towards cirri, which are complex ciliary structures of the ventral surface, either abut or bend around the cirral base. MT adjacent to or closely associated with the ciliary feeding structures (membranelies of the adoral zone of membranelies or AZM) are oriented parallel to the long axis of the AZM. Some of the MT within the oral cavity have quite complex paths. The various orientations of these subpellicular MT are discussed and evaluated for hypothetical functions of cyto-skeletal support, cell shaping and organelle movement. Each of these roles is considered to be theoretically possible.
Ultrastructural analysis of the x, X2X3O sex chromosome system during the spermatogenesis of Tegenaria domestica (arachnida)
Authors: Benavente Ricardo , Wettstein Rodolfo , Papa Mercedes
Published: April 2021

An ultrastructural study was performed on the sex chromosomes (<J XjXtXaO) during the spermatogenesis of Tegenaria domestica (Arachnids, Agelenidae). This study was carried out using random and serially cut sections. During pachytene and diplotene the three × chromosomes are longitudinally paired. Each of these consists of a central core of condensed chromatin, surrounded by a field of dense chromatin projections through which the chromosomes are in contact with one another. These projections may be responsible for the recognition and pairing of the sex chromosomes and in some way participate in their non-disjunction during anaphase I. A study of the structure and behaviour of the sex chromosomes during spermatogenesis is also presented. The available information on non-synaptonemal complex-mediated chromosome pairing and a systematization of sex chromosome structure in spiders are discussed.
Membrane lipid metabolism in Chlamydomonas reinhardtii 137+ and Y-l:: I. Biochemical localization and characterization of acyltransferase activities
Authors: Jelsema Carole L. , Michaels Allan S. , Janerox David R. , Barrnet Russell J.
Published: April 2021

The acyltransferases involved in the synthesis of the chloroplast membrane glycerolipids were analysed biochemically in dark-grown and greening Chlamydomonas reinhardtii y-1 as well as in the synchronous wild-type algae (strain 137+) and wild-type membranes. Using oleoyl-CoA as a substrate, three acyltransferase enzyme activities were detected. Glycerol-3-phosphate (glycerol-3-P) acyltransferase exhibited a pH optimum of 8·0 and was inhibited by addition of N-ethylmaleimide (MalNEt). Lysophosphatidate (PtdLys) acyltransferase exhibited a pH optimum of 7·0 and was not affected by the addition of MalNEt. From preliminary analyses, the activity at pH 5 ·5 appeared to be associated with dihydroxyacetone phosphate acyltransferase activity.

Both glycerol-3-P and PtdLys acyltransferases were analysed further and found to be present in dark-grown and light-induced y-1 cells as well as in synchronous 137+ cells and their photo-synthetic membranes. Both enzyme activities were enriched at least 10-fold in the photosynthetic membranes of 137+ chloroplasts relative to the activities present in the whole cells. This enrichment is indicative of their intrinsic localization in the thylakoids, suggesting that the photosynthetic membranes exhibit a greater degree of autonomy with respect to the synthesis of their membrane lipids than previously reported.

A role for glycerol-3-P and PtdLys acyltransferases in the synthesis of the chloroplast membrane lipids is suggested further by the increases in both enzyme activities coincident with and preceding thylakoid biogenesis following light induction of dark-grown y-1 cells. Increased acyltransferase activity preceded the increase in the chlorophyll content of greening y-t cells, which is a generally accepted marker for thylakoid synthesis. The increase in the PtdLys acyltransferase activity upon light-induclion of the y-1 cells was both more immediate and more dramatic than the increase in glycerol-3-P acyltransferase activity. PtdLys acyltransferase activity was negligible in dark-grown cells and the dramatic increase upon light induction may be important in the subsequent initiation of chloroplast membrane lipid synthesis. On the basis of the localization of acyltransferase enzyme activities to the photosynthetic membranes of 137 + cells and the increase in acyl transferase activity both preceding and occurring in concert with thylakoid synthesis, we propose a direct role for the photosynthetic membranes in the synthesis of their membrane lipid components.
Chinese hamster cells can be reversibly blocked before mitosis with the protein synthesis inhibitor, emetine
Authors: Westwood J. Timothy , Wagenaar Emile B.
Published: April 2021

The inhibition of protein synthesis in eukaryotic cells will prevent them from entering mitosis. Emetine inhibits peptide elongation. When it was added to asynchronous populations of Chinese hamster ovary (CHO) cells, the mitotic index decreased sharply 30 to 40 min later. It was found that the inhibitory effect of emetine could be reversed when it was removed and the reversibility was dependent on both the initial concentration of emetine and the pH of the medium. Cell populations that were blocked by emetine for up to 2 h showed a four-to fivefold increase in mitotic index approximately 1 h after the emetine was removed. These results indicate that there is a point or period in G2 phase at which critical ‘mitotic proteins’ are being synthesized, and if their synthesis is interrupted cells will fail to enter mitosis.
Ultrastructural studies relating to the surface morphology of cultured cells
Authors: Pugh-Humphreys R. G. P. , Sinclair W.
Published: April 2021

Scanning electron microscopy and a surface-replica technique for transmission electron microscopy have been used to study the ultrastructural features of cultured-cell surfaces The presence of microvilh measuring 0·1–0·2 μm in diameter by up to 5 μm in length has been noted as a regular feature of Landschutz ascites, ‘fibroblastic’ HeLa, and canine kidney, cells. The surfaces of chick mesenchyme cells were notably almost devoid of microvilh. The presence of microvilh at the cell surface is discussed briefly.
The proliferation of myofibrils during muscle fibre growth
Authors: Goldspink G.
Published: April 2021

Myofibrils in muscle fibres of different sizes and different ages were examined and measured using phase-contrast and electron microscopy. During the post-natal growth of the mouse biceps brachii muscle the number of myofibnls in some fibres increases from about 75 to 1200 The range of myofibnl size was from 0·4–12 μm. The distribution of myofibnl sizes in muscles of all ages studied was bimodal

A high incidence of longitudinal splitting of myofibrils was observed with the electron micro scope in differentiating muscle fibres and in some medium and large muscle fibres. Size measurements with the electron microscope showed that the splitting myofibrils were about twice as large as non-splitting myofibrils and that the myofibrils split more or less down the middle. A possible explanation for the splitting is that the peripheral I filaments are pulled at an angle slightly oblique to the myofibnl axis, because of the discrepancy in the A and I-filament lattice spacings. When the myofibnl reaches a certain size the oblique pull of the peripheral I filaments is strong enough to cause the Z disks to rip.

From data on the size, shape and number of myofibnls at different stages of growth it was concluded that longitudinal splitting is the means by which the number of myofibrils increases during post-natal growth.
Enlarged synaptic vesicles as an early sign of secondary degeneration in the optic nerve terminals of the pigeon
Authors: Cuénod M. , Sandri Clara , Akert K.
Published: April 2021

The terminal degeneration of retino-tectal fibres was studied electron microscopically in the pigeon Synaptic vesicles seem to undergo systematic changes which can best be observed in aldehyde-fixed material. Initially (i.e. within 12–24 h) the vesicles begin to swell. The enlargement is clearly visible after 4 days (40 % increase in diameter) and reaches a maximum at 14 days (100% increase). At the latter stage, the enlargement is almost invariably associated with the well known opacity of degenerating terminals. In contrast, normal control tissue contains nerve endings with only a few enlarged and no ballooning vesicles. The conclusion seems warranted that the ballooning of synaptic vesicles is an early sign of terminal degeneration, it appears to precede vesicular disintegration.
Control points in Neurospora crassa nuclear division cycle: Different effects of the inhibition of protein accumulation
Authors: Martegani Enzo , Tomé Franca , Trezzi Federica
Published: April 2021

The correlation between protein synthesis and the nuclear division cycle in Neurospora crassa hyphae was studied by inhibiting protein accumulation by two different experimental procedures: (1) starvation for lysine in a lysine-requiring mutant (lys-); and (2) addition of cycloheximide.

Lysine starvation in a lys-1 strain of N. crassa quickly blocked the nuclear division cycle and nuclei accumulated in Gi phase, as judged by their DNA content. After re-addition of lysine to starved cultures, a discontinuous pattern of uridine incorporation into DNA can be seen, showing that the nuclei were well synchronized. On the other hand, treatment with cycloheximide caused the arrest of a large proportion of the nuclei, also, in the G2 phase of the cell cycle. These results indicate that inhibition of protein synthesis may have multiple effects on the cell cycle in N. crassa and that, while moderate inhibition specifically blocks nuclei at a regulatory point in late G1, strong or complete inhibition demonstrates requirement for protein synthesis at other points in the cycle that are not necessarily regulatory points.
Kinetic analysis of cell spreading: II. Substratum adhesion requirements of amniotic epithelial (FL) cells
Authors: Aplin John D. , Bardsley William G. , Niven Veronica M.
Published: April 2021

The kinetics of spreading of trypsinized FL cells on plastic or glass substrata covalently or passively coated with various proteins to make simplified model extracellular matrices have been measured. Kinetics have also been obtained in the presence and absence of serum and amniotic fluid. Data from such experiments are shown to be sigmoid and have been computer-fitted with great accuracy to 12 mathematical models discussed in the accompanying paper. A log normal distribution function is shown to give the best fit over 18 different types of experiment. For the first time, therefore, such data can be characterized quantitatively and compared. We obtain the simple parameters μ(mean time to spread) and a (standard error of the mean) and show that the cells spread most rapidly in amniotic fluid or on fibronectin ‘carpets’. They also spread rapidly on a fibronectin-fibrinogen complex extracted from placenta. Spreading is slower in serum, in amniotic fluid lacking fibronectin and on type I collagen or cellular microexudate from FL cells or fibroblasts. On concanavalin A, spreading is rapid but the distribution of times to spread (as expressed by cr) is relatively wide.
Immunofluorescence microscopy of the flagella and multilayered structure in two mosses, Sphagnum palustre L. and Polytrichum juniperinum Hedw
Authors: Miller C. C. J. , Duckett J. G. , Sheterline P. , Carothers Z. B.
Published: April 2021

Antibody against tubulin from porcine brain was used to examine the distribution of tubulin in developing spermatids of Poly trichum and mature spermatozoids of Sphagnum. Cells were prepared for indirect immunofluorescence microscopy after fixation in buffered paraformaldehyde and brief incubation in cellulase. Pretreatment with cold methanol resulted in considerably enhanced immunofluorescence but exposure to Triton X-100, with or without sonication, had no effect. The antibody showed similar immunological cross-reactivity with the flagella (both basal bodies and axonemes) and the spline microtubules of the multilayered structure. This is the first direct evidence that this rigid array of stable cytoskeletal microtubules consists of tubulin. Particularly intense fluorescence from the lamellar strata of the MLS in developing spermatids provides strong support for the notion that the lamellae comprise a highly structured microtubule organizing centre (MTOC), responsible for the ordered assembly of the overlying spline tubules. The demonstration of immunological cross-reactivity with antitubulin from porcine brain tubulin, within a plant structure other than fully formed microtubules, suggests that immunocytochemistry may have considerable potential for the detection of other MTOCs. By contrast, no detectable fluorescence emanated from the granular matrix cementing the flagellar basal bodies to the spline or the spindle-shaped sheath of fibres present in the spermatozoids of Sphagnum. Disruption of the mature gametes by sonication and treatment with Triton X-100 reveals the presence of particularly strong links between the spline and subjacent nuclear envelope.
Insect intercellular junctions: rapid freezing by jet propane
Authors: Swales Lesley S. , Lane Nancy J.
Published: April 2021

The ventral nerve cord of the cockroach, Periplaneta americana, and that of the locust, Schis-tocerca gregaria, have been studied after rapid freezing by cryo-jet, using liquid propane. Such tissues, unfixed and uncryoprotected, have been compared with unfixed cryoprotected tissues, as well as with material fixed with glutaraldehyde and cryoprotected with glycerol or polyvinyl-pyrrolydone. The perineurial tight junctions in the cryo-jet-frozen tissues exhibit characteristic intramembranous P-face ridges, but frequently these are composed of smooth-surfaced strands, comparable to those seen with fast-freezing in mammalian tissues, rather than of bead-like fibres. The intramembranous PF ridges, characteristic of axonal and glial processes in the insect central nervous system, also display a smooth surface in rapidly frozen preparations. Prior fixation and/or cryoprotection produces a bead-like appearance in the ridges. The interglial gap junctions, after fast-freezing, exhibit both clustered connexon arrays in the E-face and loosely aggregated ones; hence the coupled state cannot be unequivocally associated with the latter configuration. The septate junctions between glial cells are unchanged after rapid freezing, exhibiting the typical rows of P-face intramembranous particles with complementary E-face pits that are found in replicas from fixed and cryoprotected tissues. The surfaces of the axons and glial processes exhibit pleiomorphic depressions and associated particles as well as PF pits with complementary EF mounds, both with associated I MPs. These structures are not usually seen after fixation or cryoprotection and may represent some kind of receptor structure, or axo-glial specialization.
Isolation and Characterization of invertebrate smooth septate junctions
Authors: Green C. R. , Noirot-Timothée C. , Noirot C.
Published: April 2021

Using modifications of techniques used for the isolation of macula type intercellular junctions (gap junctions and desmosomes) the arthropod smooth septate junction has been isolated from insect midgut tissue. Midguts from cockroaches or mealworms were used and membrane fractions were obtained by sucrose gradient and ultracentrifugation techniques. Preparations with reasonable concentrations of septate junction were obtained and have been studied by thin-section, negativestain and freeze-fracture electron microscopy. The junctions appeared to be well preserved, although there was evidence that the junction strands were able to slide within the plane of the membrane. Septa were seen to have a cross-striated appearance when viewed after negative staining but their exact structure remained difficult to determine.

Polyacrylamide gel electrophoretic studies demonstrated the reproducibility of the isolation procedure and showed that septa may have a 47 000 molecular weight glycoprotein component. Gel electrophoresis also gave some indication of the intramembrane biochemistry of the smooth septate junction, with proteins of 31 000 and 32000 molecular weight always occurring in the junction fractions. The junctions were, however, very sensitive to both mechanical and chemical treatments, the septa were destroyed by tough homogenization or by treatment with urea at a concentration as low as 1 M. Freeze-fracture of untreated, isolated junctions demonstrated no differences from junctions in intact tissue, while replicas of urea-treated material were more difficult to interpret as the component parts of the junctions became separated once the septa had been destroyed.

Gap junctions were also obtained and resisted both mechanical and chemical treatments, which destroyed the septate junctions. Their major protein component appeared to have a molecular weight of 36 000. Attempts to isolate pleated septate junctions (from insects, molluscs and annelids) by the same techniques failed, implying a significant difference in the structures of the two types of septate junction.
Morphology of hyaluronidase-sensitive cell coats as seen in the SEM after freeze-drying
Authors: Bard J. B. L. , Mcbride W. H. , Ross A. R.
Published: April 2021

Many adherent cells in vitro are surrounded by a transparent exclusion zone or halo, several pm thick, which red blood cells, bacteria and carbon particles cannot penetrate. This halo is rapidly and specifically removed by hyaluronidase and its high degree of hydration is demonstrated by the fact that, although fixation does not eliminate the halo, solvent dehydration does. This latter observation means that the halo cannot be visualized by conventional electron microscopic techniques. We report here that the exclusion-zone material can, however, be seen in the scanning electron microscope if cells are fixed and frozen rapidly and then freeze-dried. Many cells in cultures from a murine fibrosarcoma or from human embryonic lung treated in this way appear to be covered by a matrix that obscures the microvilli that are visible on critical-point-dried or hyaluronidase-treated, freeze-dried cells. Only where the coat is, for some reason, missing can microvilli be seen on freezedried cells. The coat structure varies from amorphous to an assembly of fine fibres ∼100 nm in diameter and its appearance is very similar to that of small drops of hyaluronic acid (10−5μgml-1) treated in the same way. Halo material is fragile and detaches itself from the cell surface within an hour of fixation. These observations suggest that the halo phenomenon reflects only the production of extracellular matrix and its turnover. The fragility of the haloes implies that, if they do exist in vivo, they are unlikely to play any structural role. The results suggest that the technique will yield information on other highly hydrated, unstable structures.
Organization of actin cytoskeleton during early endothelial regeneration in vitro
Authors: Gabbiani Giulio , Gabbiani Françoise , Heimark Ronald L. , Schwartz Stephen M.
Published: April 2021

The pattern of early cell movement after an experimental ‘wound’ and the organization of actin in stationary and moving cultured endothelial cells have been studied by means of: (1) time-lapse photography; (2) indirect immunofluorescence using anti-actin antibodies with and without pretreatment with the actin destabilizing factor present in human plasma; and (3) differential centrifugation and densitometric analysis of stained sodium dodecylsulphate/polyacrylamide gels in order to evaluate the total and relative amounts of G and F-actin. Up to 5 h after a single scratch, movement consists of a coordinate spreading and translocation of a band of about 10 cells from the wound edge. Compared to stationary cells, moving endothelial cells show: (1) no significant changes in the intensity and distribution of immunofluorescent staining with anti-actin antibodies, but an increased sensitivity of cytoplasmic actin, including stress fibres, to the actin-destabilizing factor purified from human plasma; and (2) no significant change in the total amount of actin, but a decreased relative amount of F-actin and a corresponding increased relative amount of G-actin. We conclude that endothelial cell movement in vitro is accompanied by a rapid change in the state of actin organization characterized by an overall decrease in cytoplasmic F-actin.
Three-Dimensional Geometry Of Motile Hamster Spermatozoa
Authors: Woolley D. M. , Osborn I. W.
Published: April 2021

Micrographs were made of free-swimming hamster spermatozoa using a high intensity xenon flash and two-colour darkground illumination - conditions that permit three-dimensional reconstruction of the instantaneous shape of the flagellum. From the waveforms observed, we constructed an account of the three-dimensional kinematics of this flagellum. We found that near-planar bends grew on the proximal 25% of the flagellum to reach a mean angle of 1·7 rad. Principal bends achieved a greater angle than reverse bends. As bends propagated they maintained their near-planarity and their angle, but decreased in radius of curvature. However, the plane of the more distal bends tended to become displaced, as though a predominantly clockwise torsion of the axoneme was developing in the interbend segment. This gave rise to a complex shape resembling a sinistral helix of reducing pitch and eccentricity. There are clear indications that not all cycles of bending lead to the same degree of three-dimensionality.
Organization Of Extracellular Matrix By Chick Embryonic Corneal Epithelial Cells In Culture And The Role Of Fibronectin In Adhesion
Authors: Mattey D. L. , Garrod D. R.
Published: April 2021

Corneal epithelial cells from IS-day chick embryos produce an extensive extracellular matrix when cultured for up to 7 days on plastic, glass, and gelatin-coated substrata. lmmunofluorescence studies revealed the presence of a fibrillar fibronectin matrix that became more extensive with time in culture. When cells were cultured in fibronectin-depleted medium little or no fibronectin was evident in the first 24 h, although the cells attached and spread normally when compared with controls. Considerably more fibronectin was associated with the cells after 48 h. Incubation of cells with [35S]methionine, followed by immunoprecipitation of cell extracts by specific anti-fibronectin antibody, confirmed that the cells synthesize fibronectin.

Cells cultured in fibronectin-coated substrata were able to reorganize the fibronectin into fibrillar form as well as to endocytose fibronectin. It is suggested that cells ‘comb’ fibronectin from the substratum and organize it into fibrils.

Interference reflection microscopy revealed no simple correlation between the distribution of fibronectin and focal contacts. However, some focal contacts seemed to lack fibronectin. In the presence of anti-fibronectin immunoglobulin G cells initially attached but failed to spread, and subsequently detached from glass and fibronectin-coated substrata. However, they attached and spread on collagen substrata. The results suggest that fibronectin is unnecessary for initial cell attachment but may be important in maintaining cell adhesion and spreading on certain substrata. However, it is not necessary for attachment and spreading on collagen. Cells on collagen may attach and spread directly on the substratum via specific collagen receptors or by using some other type of attachment factor(s).

Electron microscopy revealed a densely staining fibrillar matrix between the basal cell layer and substratum as well as in the intercellular spaces between cells. Components of this matrix and the cell surface were strongly stained by ruthenium red, which indicates the presence of acidic groups such as glycosaminoglycans.
Sperm chemotaxis in siphonophores: II. calcium-dependent asymmetrical movement of spermatozoa induced by the attractant
Authors: Cosson Marie Paule , Carré Danièle , Cosson Jacky
Published: April 2021

Spermatozoa from siphonophores have been shown to be attracted towards an extracellular structure, the cupule, which covers the predetermined site of fertilization of the egg.

Observations on sperm behaviour during the chemotactic response show that spermatozoa describe trajectories of large diameter (700-1000 µm) while far from the cupule, and of smaller diameter (200µm) in the cupule area. The transition between the two types of swimming occurs progressively when spermatozoa cross a 3 mm wide area around the cupule. After a few minutes 99 % of the spermatozoa keep swimming around the attractant source, following circular paths 150-200 µm in diameter.

In the absence of the attractant, comparable modifications of sperm trajectories are observed in the presence of the ionophore A23187 and high calcium concentrations. In the presence of 10−2M calcium ions, A23187-treated spermatozoa describe trajectories 200 µm in diameter, which increase up to 800 µm at lower calcium concentrations (10−6M).

In the absence of calcium ions, spermatozoa swim across the cupule area without modification of their trajectories and no sperm accumulation can be detected. This requirement of the chemotactic response for calcium ions is observed either with fresh cupules stuck on the eggs, with cupules separated from the eggs, or with cupule extracts. Moreover, a soluble component fractionated from the cupule induces, when diluted in sea water, a reduction in the size of the sperm trajectories and this also requires calcium ions.

The present data show that the chemotactic response of siphonophore sperm, which requires millimolar concentrations of calcium ions, occurs through a non-transient induction of increased asymmetry of the flagellar waveform. It is proposed that the natural attractant operates to produce an increase in the intraaxonemal calcium concentration.
Appearance of specific antigenic proteins in the maturing sexual organs of Sinapis flowers
Authors: Jacqmard A. , Lyndon R. F. , Salmon J.
Published: April 2021

Three proteins specific to the flowering state were found in Sinapis by immunological techniques. Two of these are specific to the stamen and one to the pistil. By the use of a histoimmunofluorescence technique their localization in the developing flower primordia and in the apex was examined during the transition to flowering. These proteins are not detected in the apex at evocation. They all appear at a relatively late stage of stamen or pistil maturation. The stamen proteins are localized in both the intine and exine layers of pollen grains in stamens 2-3 mm long; at anthesis they are essentially in the exine. The pistil protein is found in the stigma and in the transmitting tissue of the style. All these proteins contain sugar residues. A possible implication of these proteins in the process of male-female recognition is discussed.
Control of cellular differentiation by temperature in the cellular slime mould Dictyostelium discoideum
Authors: Maeda Mineko
Published: April 2021

The effects of low temperature on morphogenesis and cellular differentiation of Dictyostelium discoideum were examined. During incubation at 5 °C, the vegetative and preaggregation cells never developed, but cell masses at the aggregation or slug stage developed to form hemispherical, or dumbbell-shaped multicellular structures. By staining with FITC-antispore IgG, the structures formed after 10 days of incubation of tipped aggregates at 5 °C were found to be composed of 90% spores, 5% prespore cells and 5% non-stained cells. Since only 20% of the total cells constituting the tipped aggregate had been prespore cells at the beginning of incubation, this showed that spore differentiation proceeded even at low temperature, while stalk differentiation was completely inhibited. Similar results were obtained when the cells were incubated at 3 °C. However, at 0°C, morphogenesis and cellular differentiation did not occur, although most of the prespore cells at the late culmination stage differentiated incompletely into spores. Possible reasons for the high proportion of spores being induced by low temperature are discussed.
Mechanical stretching increases the number of epithelial cells synthesizing dna in culture
Authors: Brunette D. M.
Published: April 2021

The influence of mechanical stretching on epithelial (E) cells was examined by culturing E cells derived from the epithelial cell rests of Malassez on a flexible plastic substrate and stretching the substrate by means of an orthodontic screw. A significant increase in the number of E cells synthesizing DNA was observed after just 30 min of stretching. In 17 experiments the ratio of cells labelled with tritiated thymidine in cultures stretched for 2h to the number of labelled cells in unstretched controls was 1·92 ± 0·34. An increase in labelling as a result of stretching was found for E cells cultured at either high or low cell-population densities but the effect was most pronounced for E cells cultured at higher concentrations of foetal bovine serum. Morphometric analysis of electron micrographs of stretched and unstretched cultures indicated that the stretched cultures had a higher volume fraction of filamentous structures and more desmosomes per unit length of cell membrane than unstretched cultures. The behaviour of E cells in response to stretching in vitro appears to be similar to the response of the epithelial rests in vivo when the latter are exposed to tension as a result of forces produced by orthodontic techniques.