bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of the chloroplast and its dna in chloromonadophycean algae
Authors: Coleman Annette W. , Heywood Peter
Published: April 2021

The arrangement and ultrastructure of chloroplasts is described for the Chloromonadophycean algae Gonyostomum semen Diesing and Vacuolaria virescens Cienkowsky. The chloroplasts are present in large numbers and are discoid structures approximately 3–4 μm in length by 2–3 μm in width. In Gonyostomum semen the chloroplasts form a single layer immediately interior to the cell membrane; frequently their longitudinal axis parallels the longitudinal axis of the cell. The chloroplasts in Vacuolaria virescens are more than 1 layer deep and do not appear to be preferentially oriented. In both organisms, chloroplast bands usually consist of 3 apposed thylakoids, although fusion and interconnections between adjacent bands frequently occur. External to the girdle band (the outermost thylakoids) is the chloroplast envelope. This is bounded by endoplasmic reticulum but there is no immediately apparent continuity between this endoplasmic reticulum and the nuclear envelope. Electron-dense spheres in the chloroplast stroma are thought to be lipid food reserve. Ring-shaped electron-translucent regions in the chloroplast contain chloroplast DNA. The DNA is distributed along this ring in an uneven fashion and, when stained, resembles a string of beads. Each plastid has 1 ring, and the ring is unbroken in the intact plastid.
Quantitative cell fusion: The fusion sensitivity (fs) potential
Authors: Röhme Dan
Published: April 2021

The dose response of Sendai virus-induced cell fusion was studied in 10 mammalian cell lines, comprising 5 continuous and 5 diploid cell lines originating from 5 species. The extent of fusion was calculated using a parameter directly proportional to the number of fusion events (t-parameter). At lower levels of fusion the dose response was found to be based on the same simple kinetic rules in all cell lines and was defined by the formula: t = FS FAU/(1 + FS. FAU), where FS (fusion sensitivity) is a cell-specific constant of the fusion rate and FAU (fusion activity units) is the virus dose. The FS potential of a cell line was determined as the linear regression coefficient of the fusion index (t/(1 — t)) on the virus dose. At higher levels of fusion, when the fusion extent reached cell-line-specific maximal levels, the dose response was not as uniform. In general, and particularly in the cases of the diploid cell lines, these maximal levels were directly proportional to the FS potentials. Thus, it was concluded that the FS potential is the basic quantitative feature, which expresses the cellular fusion efficiency. The fact that FS varied extensively between cell lines, but at the same time apparently followed certain patterns (being higher in continuous compared to diploid cell lines and being related to the species of origin of the cells), emphasizes its biological significance as well as its possible usefulness in studies of the efficiency of various molecular interactions in the cell membrane/cytoskeleton system.
Coiling of the flagellar shaft in the head region of the mature sperm of Nucella (Purpura) Lapillus (L.)
Authors: Walker Muriel
Published: April 2021

The sperm of Nucella are long and threadlike. The fiagellar shaft runs from the acrosome at the front of the head to the tip of the tail. Its anterior portion, the head shaft, is ensheathed by the nucleus. If a suspension of sperm in normal-concentration sea water is observed under phase contrast, and allowed to dry out slowly, the nuclear material of sperm near the edge of the coverglass swells and the head shafts of these sperm are thrown into gentle spirals within the nuclei. In some sperm the nuclear material disperses completely, and the front three-quarters of the head shaft springs into a tight right-handed coil of 5–7 turns. Instantaneous coiling of the head shaft may also be induced by treatment of the sperm with x 2 concentrated sea water or 0·01 % (w/v) solution of sodium lauryl sulphate in sea water. The enzymes pronase and trypsin at a concentration of 50 μg/ml in sea water at pH 8·0 cause dispersion of the head nucleoprotein and subsequently the head shaft forms a loose coil. The appearance and activity of the sperm do not change perceptibly over a pH range of 5·5—8·5.

Sperm spread on a Langmuir trough containing normal concentration sea water were negatively stained with phosphotungstic acid and examined with an electron microscope. After such treatment the nuclei are partially spread and the fibrils of the head shafts appear twisted as the wires of an electrical flex. The twisted flagellar fibrils are seen more clearly in negatively stained head shafts of sperm whose nuclei have been completely dispersed by spreading on a Langmuir trough containing × 2 concentrated sea water. Negatively stained preparations of sperm treated with enzymes show sperm with twisted and coiled head shafts. The nucleoprotein of these sperm is disaggregated into strips or sheets, or may appear as a mass of branching fibres. The flagellar fibres of the enzyme-treated sperm are often bent or broken.

Longitudinal sections of mature sperm heads show that in the intact sperm the fibres of the head shaft are not twisted but run straight throughout the length of the head. Sections from the testis and testicular duct show that microtubules are present in sperm in the testis but absent from mature sperm in the testicular duct.

It is suggested that the head shaft, as a consequence of some event in spermiogenesis, has an inherent tendency to twist and coil but in the mature sperm it is ‘strait-jacketed’ by the sperm nucleus.
The crystal lattice of the pyrenoid matrix of Prorocentrum Micans
Authors: Kowallik K.
Published: April 2021

The pyrenoid matrix of the marine dinoflagellate Prorocentrum micans is shown to consist of regular close-packed units, which form a cubic face-centred lattice. Numerous lamellae usually consisting of two apposed thylakoids traverse the pyrenoid matrix. They normally run strikingly parallel to each other, with an average distance of 139 nm between each stack. The three-layered unit membrane of the thylakoids penetrating the pyrenoid is 70Å thick, the same as the unit membrane of the chloroplast thylakoids. The total thickness of one thylakoid measures 190–220 Å.

The globular units of the pyrenoid matrix have a calculated mean diameter of 232 Å, forming different line and dot patterns (hexagonal and cubic arrays) due to different section angles. Hexagonal patterns on prints result from projections of superimposed close-packed layers; they do not belong to one close-packing. Line patterns parallel to the thylakoid direction are composed without exception of 11 contrasted lines (6 and 5 alternating). This fact suggests that a definite number of units is arranged between the thylakoid stacks, thus producing the constant matrix thickness. Comparable regions in the chloroplast matrix cannot be confused with the pyrenoid matrix, as they never display an ordered structure. A three-dimensional model of the matrix is presented.

Starch is stored only in the cytoplasm; there is no visible connexion with the pyrenoid. On the evidence of protein storage in similar crystal lattices both in plants and animals it is suggested that the pyrenoid may also be an organelle for protein storage in some groups of algae.
Observations on the fine structure and development of the spindle at mitosis and meiosis in a marine centric diatom (Lithodesmium Undulatum): II. the early meiotic stages in male gametogenesis
Authors: Manton Irene , Kowallik K. , Von Stosch H. A.
Published: April 2021

Various cytoplasmic phenomena, including spindle structure and development during prophase of the first meiotic division, are described and illustrated. The living culture is represented by a timed sequence of photographs continuing those previously published with respect to mitotic stages in the same filament. The meiotic preliminaries include the so-called swelling phase, by which the parental frustule is forced open, liberating the contained spermatocytes. This occurs during pachytene on evidence of chromosome structure which is illustrated. A spindle precursor is shown to be present before opening of the frustule; this resembles structurally the mitotic equivalent though the ground plan is oblong instead of square. Growth of the precursor continues until after opening of the frustule, when the spindle itself begins to be laid down. Two stages of developing spindles during the later prophases are illustrated by sections cut in three planes and by serial sections. Preliminary comparisons are made with metaphase I and with mitosis, both qualitatively and quantitatively, but a full discussion is deferred pending completion of the record for the later meiotic stages.
The classes of endosymbiont of Paramecium Aurelia
Authors: Beale G. H. , Jurand A. , Preer J. R.
Published: April 2021

The endosymbionts of Paramecium aurelia appear to consist of a number of different Gram-negative bacteria which have come to live within many strains of paramecia. It is not known whether in nature this relationship is mutually beneficial or not. The symbionts from one paramecium may kill other paramecia lacking that kind of symbiont. We identify the following classes of endosymbiotic organisms. First, kappa particles (found in P. aurelia, syngens 2 and 4) ordinarily contain highly characteristic refractile, or R, bodies, which are associated with the production of a toxin which kills sensitive paramecia. In certain mutants of kappa found in the laboratory both R bodies and ability to kill have been lost. Second, mu particles (in syngens 1, 2 and 8) produce the phenomenon of mate-killing. Third, lambda (syngens 4 and 8) and sigma particles (syngen 2) are very large, flagellated organisms which kill only paramecia of syngens 3, 5 and 9, and are enclosed in membrane-bound vacuoles. Fourth, gamma particles (syngen 8) are minute endosymbionts, surrounded by an additional membrane resembling endoplasmic reticulum. They have strong killing activity but no R bodies. Fifth, delta particles (syngens 1 and 6) possess a dense layer covering the outer membrane. At least one of the two known stocks is a killer. Sixth, nu particles are a heterogeneous group of particles (syngens 2 and 5) which do not kill or possess distinctive morphological characteristics. Seventh, alpha particles (syngen 2) are the only known nuclear symbionts of P. aurelia; they are found in the macronucleus. Alpha is also exceptional in being the only particle which is highly infectious, though certain of the other symbionts can also be taken up by paramecia lacking them, under special conditions.
The synthesis of deoxyribonucleic acid and nuclear histone of the X chromosome of the Rehnia Spinosus spermatocyte
Authors: Bloch D. P. , Teng Christina
Published: April 2021

The X chromosome of the Rehnia spinosus (Orthoptera) spermatocyte exists in a vesicle separate from the rest of the nucleus during its replication. This chromosome is typically heterochromatic, and late replicating. After replication the chromosome vesicle fuses with the nucleus. Cytophotometric determination of DNA and histone during replication of the chromosome revealed two types of histone. One class increases in amount in proportion to the DNA. The second class remains constant as DNA doubles, and probably increases later. Autoradiographic studies of incorporation of amino acids indicates that histone labelling occurs during chromosome replication. However, a lag in amino acid incorporation suggests that DNA replication in the X chromosome, while accompanied, or closely followed, by complexing with histone, is not necessarily coupled with its synthesis.
Movement of cytoplasmic proteins into nuclei induced to enlarge and initiate dna or RNA synthesis
Authors: Merriam R. W.
Published: April 2021

In other studies it has been shown that somatic cell nuclei, which normally do not divide, are induced to enlarge and synthesize DNA when introduced into the cytoplasm of egg cells of Xenopus laevis. Introduction of such nuclei into the cytoplasm of large oocytes, however, causes nuclei to enlarge in a different way and to synthesize RNA but not DNA.

In this study the proteins of eggs and oocytes were labelled with radioactive amino acids. Brain or blastula nuclei were then injected into cells containing labelled proteins under conditions in which protein synthesis was inhibited. Movement of cytoplasmic proteins was studied by observing the increase in acid-insoluble label over the injected nuclei by quantitative autoradiography.

To prevent nuclear protein synthesis during the experiments, puromycin was injected with the nuclei. An estimation of the size of the labelled amino acid pool, a demonstration of the inhibitory effects of puromycin, and comparison of the amount of labelled material with and without puromycin all showed that protein synthesis in the nuclei played an insignificant role during the course of the experiments.

A movement of acid-insoluble label from cytoplasm of egg cells into injected brain nuclei was noted even before they had begun to swell or synthesize DNA. In the initial period of nuclear enlargement there was a disappearance of the heterochromatic clumps characteristic of brain nuclei. This period coincided with a very rapid uptake of label to concentrations about twice that of the surrounding cytoplasm. A subsequent phase of nuclear swelling was characterized by a dilution of stainable nuclear material, loss of basophilia, and establishment of acidophilia of the nuclear contents. Cytoplasmic proteins continued to enter nuclei during this phase, but at a slower rate. Extraction of the soluble materials of labelled eggs with 0·01 M NaCl at pH 7·8 and a subsequent fractionation of the extract showed that there were many radioactive compounds present with molecular weights greater than 5000.

The synthesis of DNA was initiated even before nuclear swelling could be detected, proceeding at least through the early stages of swelling.

The induction of enlargement in blastula nuclei by oocyte cytoplasm containing labelled proteins was also accompanied by an uptake of label from the cytoplasm. In this case, although the uptake of unlabelled acidophilic material was much greater, the uptake of labelled proteins was much slower than that observed in the egg.

The results are discussed in terms of chromosomal changes which occur during nuclear enlargement and during concomitant changes in nucleic acid metabolism.
Action of colchicine and heavy water on the polymerization of microtubules in wheat root meristem
Authors: Burgess J. , Northcote D. H.
Published: April 2021

The effects of colchicine and heavy water on cell division in wheat roots have been examined by means of mitotic index measurements correlated with an electron-microscope examination of the tissue. Heavy water alone caused an arrest of mitosis both in normal tissue and in roots which had been brought into partial synchrony of division by means of 5-aminouracil. Colchicine alone caused an accumulation of cells at c-metaphase in the synchronized tissue. Colchicine dissolved in heavy water showed variable effects; at high heavy water concentrations (greater then 70 %) mitosis was arrested; when the heavy water concentration was less than 70%, the mitotic index was approximately equal to that obtained by treatment of the tissue with colchicine dissolved in ordinary water.

These actions of colchicine and heavy water on the mitosis of the cells have been related to the formation and organization of the microtubules in the dividing cells. Heavy-water treatment was characterized by an increase in the number of microtubules found in the preprophase band and in the mitotic spindle. The microtubules of the preprophase band in cells treated with heavy water were arranged in a highly disoriented manner. After colchicine treatment the numbers of microtubules at the preprophase band and in the mitotic spindle were depleted. The preprophase band consisted of a few microtubules situated close to the wall. In the mitotic spindle the microtubules were either not present or visible only in the vicinity of profiles of endopla8mic reticulum or the chromosomes. When the cells were exposed to a solution of colchicine in 70 % heavy water microtubules were absent from the mitotic spindle and the preprophase band consisted of a few microtubules which were sometimes found scattered both along the wall and dispersed further into the cytoplasm.
Observations on the central pair of microtubules from the cilia of Tetrahymena Pyriformis
Authors: Chasey D.
Published: April 2021

Frayed cilia from Tetrahymena pyriformis have been examined in the electron microscope using both uranyl acetate and phosphotungstic acid as negative stains. Observations on the central microtubules show that one member of the pair exhibits two rows of short projections. These are about 18 nm long, and are spaced longitudinally at 16-nm intervals. In many specimens the projections are separated from the tubule wall and appear as a ladder-like structure linked by one or more longitudinal filaments. These results are related to some recent work on sectioned material; it is likely that the projections are independent of any central sheath holding the two tubules together.
Mitotic delay following inhibition by 5’-fluorodeoxyuridine of S-phase in Physarum is not due to delay in termination of S-phase
Authors: Kauffman Stuart A. , Shymko Ronald M.
Published: April 2021

It has been known for several years that inhibition by 5′-fluorodeoxyuridine (FdUrd) of DNA synthesis in plasmodia of Physarum polycephalum delays subsequent nuclear mitosis. To test whether this delay is due to delay in the termination of 5-phase, we blocked DNA synthesis with FdUrd + uridine for 3 h at different stages of S-phase, and in plasmodia with different cycle times. The results show that in short-cycling plasmodia the delay in mitosis can be as long as 9 h, despite little delay in termination of S-phase, and is longest when plasmodia are blocked in early S-phase. In plasmodia with long cycle times, no mitotic delay following 3 h inhibition by FdUrd of.S’-phase is observed. Our results suggest that mitotic delay after pulses of FdUrd is not due to delay in termination of S-phase, which therefore does not appear to ‘gate’ entry into a G1 period of fixed length. The fact that delay is longest after FdUrd blocks in early S-phase suggests that normal progress through S-phase, not its termination, is critical for the timing of the subsequent mitosis. This may reflect an obligate coupling between replication and transcription of specific genes needed for progress toward mitosis. The lack of mitotic delay in long-cycling plasmodia shows that S-phase-coupled processes need not act as ‘timers’ if other processes become rate-limiting.
Effect of colcemid on the centriole cycle in chinese hamster ovary cells
Authors: Kuriyama Ryoko
Published: April 2021

The structural changes in the centrioles in Chinese hamster ovary cells were monitored by electron microscopy of whole mount preparations to investigate the effects of colcemid on the events in the centriole cycle. The population of mitotic cells increased with time of incubation with colcemid, but the arrest at mitosis by this drug was soon overcome, resulting in the formation of nuclei and a change in the shape of the cells again spreading over the substrate. The maximal mitotic index was reached every 25 h in the presence of either 0·10 or 0·91 μg/ml of colcemid. During this time, cells became multinucleated, increased greatly in size, and accumulated 8 to 10-nm filamentous bundles in the cytoplasm instead of microtubules, almost all of which had been depolymerized after exposure to colcemid.

In the cells that were continuously treated with colcemid, a pair of centrioles became disoriented and each subsequently produced a daughter centriole. However, these daughter centrioles elongated to only half their full length; many unusual figures in the centriolar pairs resulted from their proceeding normally to the phases for disorientation and nucleation for centrioles in the next cycle. Although the rate of centriole elongation and the frequency of formation of the daughter centrioles were decreased by increasing the concentration of colcemid, the disorientation of the centrioles was not disturbed by this drug. The inhibitory effect of colcemid on centriolar nucleation and elongation was found to be totally reversible; the formation and elongation of new daughter centrioles occurred again just after removal of the drug.

Prolonged treatment of cells with colcemid caused ultrastructural changes in the centrioles, such as the outgrowth of microtubules from the wall of centriolar triplets or the formation of unusual bundles of microtubules around the centrioles.
Coated vesicle-mediated transport and deposition of vitellogenic ferritin in the rapid growth phase of snail oocytes
Authors: Bottke W. , Sinha I. , Keil I.
Published: April 2021

The main yolk component in oocytes of the pulmonate freshwater snails Planorbarius cometa L. and Lymnaea stagnalis L. consists of the iron storage protein ferritin and iron-free apoferritin. Both compounds are deposited in the yolk in the form of large paracrystalloids, tubular structures and randomly dispersed particles. In addition, the plasm contains lysosomelike inclusions with depositions of haemosiderin. Haemosiderin is interpreted as the product of proteolytic degradation of ferritin. During the rapid growth phase of the oocytes vitellogenic ferritin is transported across the basement lamina and taken up by adsorptive endocytosis via coated pits and vesicles. Formation of yolk bodies occurs by fusion of ferritin-containing vacuoles and empty vesicles that are probably derived from the Golgi apparatus. Uptake of ferritin is restricted to the basal region of the oocyte. No involvement of the follicle cells in synthesis and deposition of ferritin could be detected. Secretory cells of the midgut gland are the most likely site of synthesis of vitellogenic ferritin.

Under conditions of iron overload large masses of ferritin are encountered in the basement lamina of the oocytes. However, no significant increase in the uptake of ferritin could be observed. With the use of a tannic acid-glutaraldehyde fixation procedure a hitherto unobserved filamentous or rod-like material was detected inside the lamina and in coated pits. This material is probably also taken up by the oocytes and integrated into yolk platelets.

Though ferritin is a rather unusual vitellogenic protein, the mode of its uptake and deposition in the oocyte plasm is highly reminiscent of that of typical hormone-induced vitellogenins in other animal groups.
Morphology and physiology of gamete mating and gamete fusion in the fungus Allomyces
Authors: Pommerville Jeffrey
Published: April 2021

New structural features of gametes are presented and several physiological aspects of mating and gamete fusion in the fungus Allomyces macrogynus are described. In the posterior region of the male gametes, where fusion is thought to occur, thin-section electron microscopy demonstrated the presence of microfilaments that were associated with a ruffling of the otherwise smooth plasma membrane. When the cells were treated with the microfilament-disruptive drug cytochalasin B, fusion was inhibited due to the interaction of the drug with the female gamete. Freeze-fracture replica analysis of the posterior region of the gamete plasma membrane showed an area devoid of membrane particles (termed the flagellar collar) at the basal end of the flagellum. Physiological analysis of gamete mating and fusion showed a requirement for divalent cations, and the time required for gamete mating could be lengthened or shortened by decreasing or increasing, respectively, the divalent cation concentration. Diphenhydramine and chloroquine also increased the time required for mating or completely prevented fusion. Tertiary amine local anaesthetics produced the same type of results, as did trypsin and Pronase. Phospholipases were without an inhibitory effect. When the gametes were washed free of the drugs prior to mating, it was seen that the female gametes were more sensitive to cell surface changes than the males. In addition, 2-h-old female gametes showed a 70% loss of fusion ability with young male gametes while the reverse experiment showed only a 20% decrease. These observations support the concept that gamete mating and fusion may occur at the flagellar collar area and that mating in A. macrogynus is governed, at least in part, by cation and protein interactions.
The relationship of fibroblast translocations to cell morphology and stress fibre density
Authors: Lewis L. , Verna J.-M. , Levinstone D. , Sher S. , Marek L. , Bell E.
Published: April 2021

Translocation of human fibroblasts in culture was studied using techniques of time-lapse cinemicrography, indirect immunofluorescence, and computer analysis. An inverse relationship between the velocity of cells during the last hour of life and the density of stress fibers seen by immune staining was demonstrated. Translocating cells generally assumed one of two interconvertible morphologies: a triangular tailed shape or tailed fibroblast (TF), and a tailless form that resembled a half-moon, which we call a half-moon fibroblast (HMF). The tail of TFs formed only on regions of substrate that had been previously traversed by cells. The halfmoon morphology developed either on previously used or on virgin substrate. Cells adopted the HMF rather than the TF morphology with a four-fold greater frequency. HMFs translocated slightly faster than TFs. The foregoing observations suggest that the fibroblast tail is not an organelle essential for translocation. Since our technique allowed us to distinguish between cells which were cycling and those which had left cycle, we compared their velocities and found them to be similar. Also the average velocities of cells of different population-doubling levels (10th, 30th and 40th) were approximately equal.
Fine structural changes in the fat body cells of Rhodnius (hemiptera) during extreme starvation and recovery
Authors: Wigglesworth V. B.
Published: April 2021

The fate of lipid-rich components in the fat body cells of Rhodnius during extreme starvation and recovery is described. The mitochondria (along with ER etc.) degenerate to form large cytolysomes whose amorphous contents are dispersed after feeding to yield watery vacuoles with lipofuscin in their walls. Surviving mitochondria become swollen, with reduced cristae and conspicuous deposits of residual lipid or lipofuscin. In extreme starvation chromatin is progressively dispersed and discharged from the nucleus.

After feeding, damaged mitochondria recover very slowly before multiplying ; there seems to be no new formation of mitochondria. New ER is formed within the first hour after feeding by the escape of lipid-rich material from the nucleolus, which leads to expansion of the outer nuclear membrane to form blisters; these are budded off to form cysts and their surface is evaginated repeatedly to form laminated ER. New lipid droplets in the fat body appear to arise from the condensation of clusters of lipid-rich particles which are derived from the ER.
Relation of cytoplasmic calcium to contractility in Physarum polycephalum
Authors: Kuroda Ritsu , Kuroda Hideyo
Published: April 2021

In a dumbbell-shaped plasmodium of Physarum polycephalum showing active shuttle streaming Ca was precipitated with potassium pyroantimonate (K[Sb(OH)6]), and the distribution of Ca between the cytoplasm and cellular organelles, especially vacuoles, was examined by electron microscopy. The contracting half-mass, where many empty vacuoles were present, was rich in the small Ca precipitates located in the cytoplasm. The relaxing half-mass, where many Ca-containing vacuoles were present, was poor in the cytoplasmic Ca precipitates. One half-mass of a dumbbell-shaped plasmodium was treated with Ca ionophore, X-537A, and its effect on the motive force for endoplasmic streaming and the distribution of Ca was investigated. The motive force was increased by X-537A, but the period of shuttle streaming was not changed. X-537A also induced a significant increase in the number of the cytoplasmic Ca precipitates in the X-537A-treated contracting half-mass, so that the asymmetry of the distribution of cytoplasmic Ca precipitates was enhanced. A large portion of the vacuoles were empty in the contracting half-mass, and Ca-containing in the relaxing one as in the case of the untreated plasmodium.
Effect of the ionic environment on the incorporation of the intermediate-sized filament protein vimentin into residual cell structures upon treatment of ehrlich ascites tumour cells with triton x-100: I. Biochemical analysis
Authors: Traub Peter , Nelson W. James
Published: April 2021

Ehrlich ascites tumour cells, propagated in suspension culture, were extracted with Triton X-100 under different ionic conditions to study the sensitivity of intermediate-sized filaments formed in vivo to changes in the ionic environment in vitro. The following results were obtained : (1) in solution of low ionic strength and in the absence of di- and polyvalent cations, vimentin was quantitatively solubilized and recovered from the postnuclear supernatant by precipitation with (NH4),SO4 (23% saturation) or spermidine (2 BIM). (2) When the cells were extracted in low ionic strength buffer in the presence of 4 mM-Mg-acetate or 1 · 2 mM-spermidine, vimentin was quantitatively incorporated into Triton X-100-resistant residual cell structures. It could be easily extracted from Mg2+-stabilized cell residues with Mg2+- and polyamine-free buffer of low salt concentration; spermidine-stabilized cell residues were very resistant to dissociation. (3) In a solution of high salt concentration and in the absence of di- and polyvalent cations, little or no vimentin was retained in residual cell structures. Better, though not quantitative, binding was observed in solution at physiological NaCl or KC1 concentrations. (4) A synergistic effect of mono- and di- or polyvalent cations on the association of vimentin with the nuclei was detected when the extraction of cells was performed at concentrations of these ions, which individually were very inefficient in the formation of residual cell structures. (5) Triton X-100-resistant cell residues formed under any ionic conditions were rather resistant to shearing forces. If release of vimentin occurred, it was due to perturbance of the dissociation equilibrium between bound and free vimentin by dilution or washing. (6) Preincubation of cells in buffers of varying ionic composition and in the absence of Triton X-100, followed by titration of the amount of vimentin still incorporable into residual cell structures by extraction of cells with Triton X-100 in solution of 4 mM-Mg2+, showed that the incorporation of vimentin into cell residues was strictly dependent on the intracellular ionic conditions. (7) The intracellular dissociation of vimentin-containing intermediate-sized filaments, induced by preswelling of cells in low ionic strength buffer, could be reversed, to a considerable extent, by restoration of isotonicity. If the incorporation of vimentin into Triton X-100-resistant residual cell structures is taken as a measure of the intactness of the vimentin-type intermediate-sized filaments, the experimental results demonstrate that the stability of the filaments is largely dependent on the maintenance of physiological concentrations of mono-, di- and polyvalent cations.
Fractionation of isolated liver cells after disruption with a nitrogen bomb and sonication
Authors: Autuori F. , Brunk U. , Peterson E. , Dallner G.
Published: April 2021

Hepatocytes from rat liver were prepared by perfusion with collagenase, and rough and smooth microsomes and mitochondria were prepared after cell disruption. By applying 1000 lb/in2 (1 lb/in2 = 6·9 kPa) in a nitrogen bomb followed by decompression, 75% of the cells were disrupted after four consecutive treatments. Intact mitochondria, and rough and smooth microsomes with little contamination were prepared from the homogenate. A more rapid disruption was attained by a short sonication with a low output, thus increasing the efficiency of homogenization. The microsomal subfractions prepared from this homogenate were comparable to those obtained after decompression. Sonication resulted in smooth microsomes, which exhibited a higher contamination with non-microsomal membranes. These, however, were partly removed by additional centrifugation with a discontinuous sucrose gradient containing divalent cations.
Control of histone gene expression in Physarum polycephalum : I protein synthesis during the cell cycle
Authors: Schofield P. N. , Walker I. O.
Published: April 2021

Synchronous cultures of Physarum polycephalum were pulsed with [3H] lysine hydrochloride in S and G2 phases of the cell cycle. Plasmodial extracts were separated into nuclear, ribosomal and acid-soluble post-ribosomal cytoplasmic fractions. Core histones could be detected by staining in the nuclear fractions of both S and G2 phases, but were not detected by staining in the cytoplasmic fractions. Newly synthesized histone was present in S-phase nuclei but not in S-phase cytoplasm. The specific activity of newly synthesized histone in G2-phase nuclei decreased by at least 95% compared to S phase and no newly synthesized histone was observed in G2-phase cytoplasmic fractions. Thus histone synthesis is restricted to 5 phase. There are no free pools of histone in the cytoplasm of Physarum in either S or G2 phases of the cell cycle.
Epidermal cell migration on collagen and collagen-derived peptides
Authors: Donaldson Donald J. , Smith Gerald N. , Kang Andrew H.
Published: April 2021

Nucleopore filters coated with various genetic types of collagen and certain collagen-derived peptides were implanted under one margin of a skin wound on adult Notophthalmus viridescens (newt) hind limbs. In contrast to their behaviour on untreated filters, epidermal cells migrated readily and to equal degrees on human type I, newt type I, bovine type II, and bovine type IV collagen. Denaturation had no effect on the ability of collagen to support migration and all three cyanogen bromide peptides tested (α1 (I)CB3, 7 and 8) were able to support more migration than that seen on untreated filters. Glutaraldehyde-linked collagen gels supported migration but bovine serum albumin gels did not.

These results show that there is no species or collagen-type specificity shown by newt epidermal cells as they migrate over collagen-coated substrates. They also demonstrate that the tertiary structure of the collagen molecule is unimportant in its ability to bind to newt epidermal cells, and that the α1 (I) chain has at least three, and probably many epidermal binding sites. Finally, they indicate that the improved migration on collagen is not a non-specific response to protein on the substrate.
Chromatin from the unicellular red algaPorphyridiumhas a nucleosome structure
Authors: Barnes K. L. , Craigie R. A. , Cattini P. A. , Cavalier-Smith T.
Published: April 2021

We have isolated a crude nuclear preparation from the unicellular red alga Porphyridium aerugineum and investigated the structure of Porphyridium chromatin.

Electrophoresis of deproteinized DNA fragments produced by micrococcal nuclease digestion of Porphyridium nuclei gives a typical ladder pattern, indicative of a repeating structure. The DNA repeat-length, calculated from plots of multimer length against multimer number, varies somewhat between different digestions, ranging from 160 to 180 base-pairs (average 173). We interpret this as evidence of heterogeneity in repeat-length; the calculated repeat-length depends on the extent of digestion because chromatin sub-populations with longer repeatlengths are on average digested earlier.

Polyacrylamide / sodium dodecyl sulphate gel electrophoresis of basic proteins purified from Porphyridium nuclear preparations gives a pattern characteristic of core histones. Although our interpretation is complicated by some degradation, the result strongly suggests that Porphyridium chromatin contains each of the four core histones and that they are similar to those of higher eukaryotes. This, together with the micrococcal nuclease digestion results, demonstrates that Porphyridium chromatin is not fundamentally different from that of higher eukaryotes.
Ultrastructural and immunocytochemical demonstration of gametophytic proteins in the pollen tube wall of the primitive gymnosperm Cycas
Authors: Pettitt J. M.
Published: April 2021

Ripe pollen of the primitive gymnosperm Cycas armstrongii germinates 8–15 days after sowing on a suitable artificial medium. During the pre-germination interval, the tube cell of the tricellular male gametophyte is actively engaged in the synthesis and export of polysaccharide material. This polysaccharide is laid down as a continuous microfibrillar stratum between the tube cell plasmalemma and the pollen wall intine. While the polysaccharide layer is forming, numerous evaginations arise as cytoplasmic extensions from the tube cell and penetrate the component. The structural link between the cell and the evaginations is eventually lost and the isolated fragments of cytoplasm immediately break up to form discrete particles. These particles remain within the channels previously occupied by the evaginations.

At germination, the exine and intine layers of the pollen wall are disrupted and the tube cell emerges through the germinal aperture to establish the pollen tube. Polysaccharide deposition is resumed at about this stage, and consequently, a second microfibrillar layer begins to form beneath the first. The wall of the early pollen tube, therefore, has two distinct zones: the outer stratum is a loose superficial reticulum of fibrils and contains cytoplasmic inclusions; the inner stratum is structurally compact and is without inclusions.

Throughout the period of tube development, the presence of protein, including the enzyme acid phosphatase, can be demonstrated in the outer wall zone with cytochemical methods. Immunofluorescent staining using pollen antiserum immunoglobulin G in an indirect technique with fluorescein isothyocyanate-labelled secondary antibody shows that one component in the pollen tube wall is immunologically related to the male gametophyte, and results from immunoelectron microscopy support the conclusion that this consists of the inclusions rather than the polysaccharide fabric of the tube wall itself.

There are remarkable similarities between the cytoplasmic inclusions in the tube wall in Cycas and those found in the intine layer of flowering plant pollen. Both are of haplophase derivation, although the inclusions in the flowering plants are introduced via evaginations into the intine, the incipient pollen tube wall, at a much earlier stage in pollen development than they are in Cycas. The difference in the time of incorporation represents, perhaps, one more episode in the progressive evolutionary trend towards rapid pollen tube initiation and growth, and hence rapid fertilization, in spermatophyte pollen. This possibility is discussed, taking into account the view that the cycads and flowering plants are groups with a common ancestor.
Nucleic acid synthesis in microsporocytes of Lilium cv. cinnabar: events in the nucleus
Authors: Porter E. K. , Bird M.J. , Dickinson H. G.
Published: April 2021

In an electron microscopic autoradiographic study of DNA and RNA synthesis during meiosis isolated Lilium microsporocytes were supplied with [3H]thymidine and [3H]uridine. DNA synthesis occurred in the nucleus during the zygotene and pachytene intervals of meiotic prophase. Most of the activity was associated with the chromatin, but some synthesis early in zygotene was located at the nucleolus. RNA synthesis occurred throughout prophase until diplotene, when all activity ceased until after division. The newly synthesized RNA was found mostly in association with the chromosomal peripheries or in the space between chromosomes. There was also a peak of [3 H] uridine incorporation at the nucleolus, which followed shortly after the synthesis of DNA at that site. The localization of DNA and RNA synthesis at the various stages of meiosis is discussed in relation to current concepts of chromosome pairing, crossing-over, ribosomal DNA amplification and cycles of RNA metabolism.
Cell-cycle kinetics and ultraviolet light survival in uv-1, a chinese hamster ovary cell mutant defective in post-replication recovery
Authors: Collins Andrew , Waldren Charles
Published: April 2021

UV-1, an ultraviolet-sensitive mutant of CHO-K1, is abnormally slow to recover from the inhibition of DNA synthesis caused by u.v. irradiation. When synchronized UV-1 cells are irradiated in G1 their movement into S phase is unaltered, but thymidine incorporation is depressed (compared with that in the parent cell similarly treated). When irradiated in S phase, again incorporation is more depressed, and S phase suffers a greater delay in UV-1 than in the parent cell. UV-1 and its parent have similar capacities for excision repair of u.v.-induced damage inflicted in G 1 and so enter S phase with similar amounts of unrepaired damage. The single-cell survival was measured after irradiation at different points in the cell cycle. The mutant and parent cells have similar values of D0 (mean lethal dose) except in mitosis, when the parent cell shows markedly greater resistance to u.v. irradiation. Dq (quasi-threshold dose) is fairly constant for the parent cell, but in UV-1 it falls to a minimum in S phase. The responses of UV-1 to u.v. irradiation are generally consistent with its known defect in the process of post-replication recovery, i.e. the ability to join up the abnormally small DNA fragments synthesized on a u.v.-damaged template.
Nucleolar organizer structure and activity in a nucleolus without fibrillar centres: the nucleolus in an established Drosophila cell line
Authors: Knibiehler B. , Mirre C. , Rosset R.
Published: April 2021

Classical electron-microscopic techniques (enzymic digestion, EDTA regressive staining) allied with autoradiographic studies after [3H]uridine incorporation or after RNA synthesis initiated by an exogeneous RNA polymerase in the presence of tritiated GTP, enabled us to describe the fine structure and activity of the nucleolus in an established Drosophila cell line. This nucleolus is composed of a large central multilobed core containing proteins, RNA molecules and a DNA-containing component. This core is surrounded by and connected to large clumps of dense fibrillar nucleolus-associated chromatin, which are intermingled with fibrillogranular ramifications extending from the core towards the nuclear envelope. These ramifications are covered by granules of ribosomal ribonucleoprotein. As shown by EDTA regressive staining the nucleolar core contains a ribonucleoprotein network, which unravels and ramifies within a fibrous matrix. RNA synthesis takes place at the level of this network in the internal part of the core. The molecules synthesized are associated with proteins and are exported out of the core in the form of granules. Although it is composed of the same constituents as other nucleoli, the nucleolus of Drosophila cells seems to be less organized, in that it never displays fibrillar centres, which have been referred to as the nucleolar counterparts of the nucleolus-organizers in a wide variety of organisms.
The cytoskeleton and substratum adhesion in chick embryonic corneal epithelial cells
Authors: Billig D. , Nicol A. , Mcginty R. , Cowin P. , Morgan J. , Garrod D.
Published: April 2021

The adhesions and cytoskeleton of 15-day chick embryonic corneal epithelial cells have been studied using interference reflection microscopy (IRM), electron microscopy and fluorescent staining with specific antibodies against actin, tubulin, prekeratin, fibronectin and laminin In vivo, desmosomes were the most frequent intercellular junctions and hemidesmosomes were prominent at the basal surface of the epithelium in contact with the basement membrane. The intact epithelium stained for actin, tubulin, and prekeratin and laminin, but not fibronectin, and the basement membrane for fibronectin and laminin.

In monolayer culture intercellular desmosomes were reformed after dissociation with trypsin. However, we have concentrated on cell-substratum adhesions, because the relationship between substratum adhesions and the cytoskeleton has not been thoroughly investigated for epithelial cells. Most of our results apply to contacts with gelatin substrata but the situation was similar on glass or tissue-culture plastic. By IRM, focal contacts similar to those of fibroblasts were present, mainly beneath the leading lamellae of the peripheral cells of monolayered islands. Fluorescent antibody staining revealed that each focal contact was positioned at the end of an actin microfilament bundle. However, there was no correspondence between focal contacts and either prekeratin filaments or microtubules.

Fibronectin fibrils were found principally beneath peripheral cells, but evidence is presented suggesting that fibronectin is not directly involved in cell-substratum adhesion of these cells. We suggest that the fibrillar fibronectin pattern arises because cells physically reorganize fibronectin which adsorbs or binds to the substratum from the medium.

Electron microscopy suggested that these cells formed two types of contacts with the substratum : small dense plaque-like structures, probably hemidesmosomes, were present in addition to focal contacts. The former were associated with tonofilaments and were the same size (about 0 · 1 μ m in diameter) as the desmosomes and hemidesmosomes found in vivo. They were much smaller than focal contacts which were from one to several micrometers in length. These structures were not as well formed as the hemidesmosomes found in vivo, possibly because the substratum was not entirely appropriate. The significance of the two types of cell-substratum adhesions is discussed.
Ultrastructural changes in the surface layers of the newt’s egg in relation to the mechanism of its cleavage
Authors: Selman G. G. , Perry M. M.
Published: April 2021

The surface and cortical layers of an uncleaved newt egg have a characteristic ultrastructure which remains unaltered during cleavage; ultrastructural changes are confined to the region of the furrow. At the onset of cleavage there is a dipping inwards of the rough heavily pigmented animal surface to form a groove. Along the bottom of the groove the surface irregularities are reduced and a dense band (0·1 μm thick and 16μm wide) is formed immediately below the plasma membrane. Within this band there are parallel filaments, 8–10 nm in diameter, oriented in the direction of the future furrow. No structural modifications were observed below the cortical layers of the leading part of the furrow apart from accumulations of granules and the mid-bodies of the spindle remnant. It is proposed that the dipping-in of the groove is due to contraction within the filamentous band, rather than contraction in a sheet of subcortical gel as proposed previously. The filamentous band persists below the furrow during the later stages of cleavage.

The new unpigmented surface first forms as a strip across the animal surface and begins to grow at the bottom of the groove. Over most of its area, it is much smoother than the pigmented surface and has less material on the outside of the plasma membrane. There are microvilli along the bottom of the groove. The join between the new unpigmented and the old pigmented surface is abrupt. As the new unpigmented surface grows in extent, a narrow furrow forms below the lowest part of the groove and progresses towards the vegetal surface. For most of its length the furrow is between 10 nm and 0·5μm wide, but at its leading edge it is 2μm wide with microvilli on its surface and 10-nm filaments below the plasma membrane. It is concluded that the progressive formation of the furrow is due to active growth of new unpigmented cell surface.

At late cleavage a ridge 10μm high forms at the join between the new and old surface. After cleavage the ridges approach and meet to form the intercellular junction by which daughter blastomeres are held together along the animal surface.

The mechanism of cell cleavage in the newt egg and in other forms is discussed in the light of the present observations.