Ultrastructure of macrocyst formation in the cellular slime mold, Dictyostelium mucoroides: extensive phagocytosis of amoebae by a specialized cell

Macrocyst differentiation in Dictyostelium mucoroides was carried out in shaking flasks. Observations of the development of macrocysts were made by light and electron microscopy. Macrocyst development begins with the clumping of stationary phase amoebae. Subsequently, the clumps become subdivided into smaller masses, each surrounded by a fibrillar sheath. At the center of each mass there arises a cytophagic cell which proceeds to engulf in turn all the cells in the mass. The engulfed cells (endocytes) undergo drastic changes in their fine structure and eventually are transformed into granules. During the early stages of engulfment the cytophagic cell has a single large nucleus, but by the time all the cells have been converted to endocytes the cytophagic cell has become multinucleate. The multinucleate condition persists in the granular stage. The thick cellulose wall surrounding each macrocyst is produced by the cytophagic cell soon after it has engulfed all the cells in the mass and before the granular stage.     

Characterization and Solubilization of β-Glucan Synthases from Cultured Rice Cells

Two types of β-glucan synthases, GS-I and GS-II, were found in cultured rice cells (Oryza sativa L.). In glycerol density gradient centrifugation, GS-I activity peak co-migrated with a marker enzyme of the Golgi membrane, while GS-II co-migrated with the plasma membrane. Analysis of the reaction products of GS-I and GS-II, suggested that GS-I and GS-II were mainly β-1,4,- and β-1,3-glucosyltransferases, respectively. GS-I had a higher substrate affinity for UDP-glucose than GS-II, and needed divalent cations for its activity. Effects of nucleotides on the activity were also considerably different between GS-I and GS-II. GS-I was solubilized well with CHAPS and digitonin, and GS-II was solubilized effectively with sucrose monolaurate.     

Nuclear fusion in multinucleated giant cells during the sexual development of Dictyostelium discoideum

Macrocyst development in Dictyostelium discoideum, is generally considered a sexual phase. This development is initiated by the formation of a giant cell, the result of the fusion of two different mating type haploid cells, such as NC4 and HM1. The giant cell engulfs unfused surrounding cells to develop into a macrocyst. Therefore, if the macrocyst is a sexual structure, the giant cell must be a diploid zygote. However, under certain conditions, a very large multinucleated giant cell containing several dozens of nuclei is formed, followed by normal development into a macrocyst. In such a multinucleated giant cell, it was found that only two nuclei fuse together to produce a diploid zygote and all others disappear at the early stage of development. The diploid nucleus undergoes meiosis and subsequently subdivides into a number of haploid progeny cells later released from the macrocyst to initiate new life cycles.     

Isolation of a highly pure archeocyte fraction from the fresh-water spongeEphydatia fluviatilis

Summary A pure archeocyte fraction was isolated from the fresh-water spongeEphydatia fluviatilis by density gradient centrifugation of dissociated cell suspensions. The nature and purity of the fraction were confirmed by electron microscopy.     

Separation and Partial Purification of 1,3-beta-Glucan and 1,4-beta-Glucan Synthases from Saprolegnia

Enriched 1,3-β-glucan and 1,4-β-glucan synthase fractions from the fungus Saprolegnia were isolated by rate zonal centrifugation on glycerol gradient. Purification was improved by entrapment of the enzymes in their reaction product, i.e. microfibrillar glucans. 1,3-β-Glucan synthases were separated from 1,4-β-glucan synthases following resuspension of entrapped enzymes. Sodium dodecylsulfate-polyacrylamide gel electrophoresis indicated that 1,3-β-glucan and 1,4-β-glucan synthases may have a different polypeptide composition because they were enriched for different protein subunits (34, 48, and 50 kD for the 1,3-β-glucan synthase and 60 kD for the 1,4-β-glucan synthase).     

Protein Synthesis Initiated by Cell Fusion in Dictyostelium discoideum

D. discoideum has two alternative developmental pathways. If cells of two complement mating-type strains, NC4 and HM1, fuse sexually, a giant cell is produced which subsequently develops into a macrocyst, the sexual structure of this organism. However, if fusion fails to occur and cells are starved, a fruiting-body is produced instead of a macrocyst. In this paper, a two-dimensional polypeptide gel electrophoresis study showed that giant cells produce specific polypeptides which may possibly be involved in macrocyst development. Out of total 497 polypeptides which appeared in a giant cell during an incubation period of 13 hr, 92 were the specific for giant cells. Four of these polypeptides were appeared within only 1 hr after the cell fusion. The other 405 were non-specific polypeptides which appeared in both giant cells and NC4 or/and HM1 cells. However, the patterns and the rates of production of each polypeptide during the incubation period were different between these cells.     

{beta}-1,4-Glucan occurring in homogenate of Phaseolus aureus seedlings. Possible nascent stage of cellulose biosynthesis in vivo

A small amount of cytoplasmic ß-1,4-glucan, whichmight be involved in the synthesis of cellulose in the cellwall, was found in the homogenate prepared from the hypocotylsof seedlings of Phaseolus aureus. Upon hydrolysis by cellulaseof the 20,000?g pellet from the cytoplasmic fraction of segmentsincubated in a [¹⁴C]-glucose solution, [¹⁴C]-cellobiose wasproduced, with specific radioactivities 3 to 10 times greaterthan those of the cellobiose from cellulose in the cell wallat various incubation periods. The incoporation of radioactivityfrom [¹⁴C]-glucose into this cytoplasmic ß-1,4-glucanwas therefore faster than that into cellulose constituting thecell wall. Hence, it seemed that the former ß-1,4-glucancould be turned over. To examine whether the- cytoplasmic ß-1,4-glucanis carried by some subcellular components, cytoplasmic ß-1,4-glucanin the cell was fractionated by differential centrifugation,two enzyme activities being measured as the markers of subcellularcomponents. The distribution of ß-1,4-glucan was similarto that of UDPG-glucosyltransferase activity but not to thatof IDP-ase activity. The result suggests that the cytoplasmicß-1,4-glucan has some relation to plasma membranes. Coumarin, known as a specific inhibitor for the biosynthesisof cellulose in plant cells, was shown to inhibit the incorporationof radiocarbon from [¹⁴C]-glucose into cytoplasmic ß-1,4-glucanto the same extent as that into cellulose in the cell wall ofthe hypocotyls. ¹ Present address: Department of Biological Science, TohokuUniversity, Kawauchi, Sendai 980, Japan. (Received May 31, 1976; )     

IMMUNOLOCALIZATION OF AN α-1,4-GLUCAN LYASE IN YOUNG AND MATURE PARTS OF THE RED ALGA GRACILARIOPSIS SP. (RHODOPHYTA)

The enzyme α-1,4-glucan lyase (EC 4.2.2.13) was studied in cells of young and mature parts of the red alga Gracilariopsis sp. by using immunogold labeling in ultrastructural studies. In young tissues, the α-1,4-glucan lyase was observed at two different sites: around the starch granules in the cytosol and in the stroma of the chloroplast. In mature tissues, the α-1,4-glucan lyase was present only in the chloroplasts. The possible role of this starch-degrading enzyme in red algae is discussed.     

SEPARATION OF HELA CELLS BY COLLOIDAL SILICA DENSITY GRADIENT CENTRIFUGATION : I. Separation and Partial Synchrony of Mitotic Cells

Using a colloidal silica density gradient, HeLa cells in mitosis were found to have a density of 1.040–1.046 g/cc, lighter than the remaining interphase cells. The mitotic cells could be harvested and cultured after centrifugation, showing growth synchrony by measurement of a peak in mitotic index 21 hr after establishing the culture. By using Colcemid or vinblastine sulfate, HeLa cells were arrested in metaphase and centrifuged on the colloidal silica density gradient. The blocked metaphase cells were lighter in density than the interphase cells but somewhat more dense than untreated cells selected by the density gradient centrifugation. Near-equilibrium conditions were established during the centrifugation of cells so that cell density measurements could be made, and the gradient medium employed was not measurably toxic to those cells tested.     

Lectin binding and inhibition studies reveal the importance of D-glucose, D-mannose and N-acetylglucosamine during early sexual development of Dictyostelium discoideum

Fluorescein-conjugated and non-conjugated lectins were used to determine which surface sugars are involved in the early events of sexual (macrocyst) development in Dictyostelium discoideum. Only zygote giant cells showed unique binding of FITC-WGA and FITC-PNA while all cell types (amoebae, gametes, binucleates, giant cells) showed identical patterns of FITC-Con A, -Gorse and -RCA II binding. In spite of its non-selective labelling of all cell types, Con A inhibited macrocyst formation. The temporal addition of Con A with and without specific hapten sugars indicates the importance of both D-mannose and D-glucose in phagocytosis and, possibly, cell fusion. WGA also inhibited macrocyst formation. Varying the time of addition of the lectin plus/minus its primary hapten sugar implicates N-acetylglucosamine as being important in cell fusion. Neither Gorse, RCA II nor PNA had any detectable inhibitory effects on macrocyst development leaving the appearance of increased PNA receptors at the giant cell surface as an enigma.     

Sweet corn phosphorylase: purification and properties

Sweet corn 1,4-α-glucan phosphorylase was purified 190-fold to a near homogeneous state. The enzyme had a molecular weight of about 315,000 on Sephadex G-200 chromatography. The pyridoxal 5′-phosphate content was found to 1 mole per 140,000 g protein, suggesting that the enzyme is dimeric. On sucrose density gradient ultracentrifugation the sweet corn phosphorylase was dissociated to an active monomeric species with a molecular weight of 150,000 and a sedimentation coefficient of 8 S. The priming specificity of the sweet corn phosphorylase was investigated; maltose was not a primer and maltotriose was the smallest apparent primer. The Michaelis constants for the maltosaccharide series from maltopentaose to maltooctaose were determined. The effect of d-enzyme on the apparent priming specificity of the enzyme was investigated. Adenosine diphosphoglucose and 2,3-diphosphoglycerate were found to inhibit the enzyme activity.     

Density dependent growth of corneal endothelial cells cultured in vitro

Using the cornea of macaque monkey, we demonstrated the relationship between cell density and growth of endothelial cells in vitro. Corneal endothelial cells in a cell sheet grow most actively in regions with cell density of 1000 to 1800 cells/mm2, in explant cultures and cell sheets and in concentrated inocula dissociated cells. Cell morphology was well sustained in these cultures. Cells cultured at a higher cell density retained their potential to proliferate actively, showing clear contrast to cells cultured at a density lower than 200 cells/mm2. When dissociated cells were cultured at a low density and maintained for more than 4 weeks, they gradually lost their growth potential, altered into polymorphonuclear giant cells and eventually dedifferentiated. In addition, cells with no contact with each other did not express growth potential. Density dependent growth was confirmed by measuring the mitotic index against the cell density per square mm from the center to the peripheral regions in cultured explants. It is concluded that the growth pattern of corneal endothelial cells is closely related to cell density, and that growth of these cells might be regulated through intercellular communications.     

Synthesis of specific proteins for sexual development in Dictyostelium discoideum

The development of Dictyostelium discoideum may proceed by two pathways, macrocyst or fruiting-body formation, the former being the sexual and the latter the asexual cycle. The pathway of development depends on the presence or absence of zygote giant cells which are produced through fusion of opposite mating-type cells in a population, in heterothallic strains. During the early stages of macrocyst development the patterns of developmentally regulated proteins were noted to differ considerably from those during fruiting-body development. Furthermore, the haploid cells around zygote giant cells synthesized a large number of specific proteins for macrocyst development through the influence of giant cells.     

Signalling and sex in the social amoebozoans

The social amoebozoans have a life tricycle consisting of asexual multicellular development leading to fruiting bodies, sexual multicellular development resulting in macrocysts, and unicellular development generating microcysts. This review covers the events of sexual development in the best‐studied heterothallic (Dictyostelium discoideum) and homothallic (D. mucoroides) mating systems. Sexual development begins with pheromonal interactions that produce fusion‐competent cells (gametes) which undergo cell and pronuclear fusion. Calcium‐ and calmodulin‐mediated signalling mediates these early events. As they initiate chemotactic signalling, each zygote increases in size becoming a zygote giant cell. Using cyclic AMP (cAMP), the zygote chemotactically lures in amoebae and engulfs them in an act of cannibalistic phagocytosis. Chemotaxis proceeds more quickly than endocytosis because the breakdown products of cAMP (5‐AMP, adenosine) bind to a presumptive adenosine receptor to inhibit sexual phagocytosis. This slowing of phagocytosis allows amoebae to accumulate around the zygote to form a precyst aggregate. Zygote giant cells also produce several other signalling molecules that feed back to regulate early events. The amoebae surrounding the zygote seal their fate as zygotic foodstuff by secreting a primary cellulose wall, the extracellular sheath, around the zygote and aggregated amoebae, which prevents their escape. Phagocytosis within this precyst continues until all peripheral amoebae are internalized as endocytes and the final macrocyst wall is formed. Endocyte digestion results in a mature macrocyst with a uniform cytoplasm containing a diploid nucleus. After detailing the morphological events of heterothallic and homothallic mating, we review the various intercellular signalling events and other mechanisms involved in each stage. This complete and comprehensive review sets the stage for future research on the unique events that characterize sex in the social amoebozoans.     

Fusion of cell ghosts with sexually opposite type cells in Dictyostelium discoideum

In the sexual cycle of Dictyostelium discoideum, haploid cells of two opposite mating types, strains HM1 and NC4, acquire fusion-competence under certain conditions, such as suspension culture in the dark, and fuse specifically to form giant zygote cells. Each giant cell engulfs the surrounding cells, gradually increases in size, and finally develops into a macrocyst that is a sexual structure in D. discoideum. Fusion-competent HM1 cells suspended in a solution were frozen and thawed to make cell ghosts. When cell ghosts were introduced into fusion-competent and -incompetent intact NC4 cells, the cell ghosts killed them in a short time, but the fusion-competent cells were killed in preference to the fusion-incompetent cells. This killing occurred through the fusion of the cell ghosts directly to intact cell membranes. Since the fusion was specific, the fusion between ghosts and cells appears to be essentially the same as that between intact cells during the sexual cycle in molecular mechanisms.     

Validation of a multiparameter flow cytometry method for the determination of phosphorylated extracellular-signal-regulated kinase and DNA in circulating tumor cells

A simple, selective, and sensitive multiparameter fluorescence activated cell sorting method utilizing density gradient centrifugation and magnetic antibody cell sorting was developed and validated for the determination of phosphorylated extracellular-signal-regulated kinase (pERK) and DNA in circulating tumor cells (CTCs). Cell preparation tubes (CPT) were used for peripheral blood collection and density gradient centrifugation, followed by phosphorylation of ERK with epidermal growth factor (EGF). After fixation with formaldehyde and methanol, magnetic anti epithelial cell adhesion molecule (EpCAM) micro-beads were used for the selective isolation of CTCs from the background, consisting of peripheral blood mononuclear cells and platelets. Subsequently, samples were stained with Hoechst 33342, and fluorescent antibodies against EpCAM, CD45, and pERK. Flow cytometry was used for identification and enumeration of CTCs and determination of their pERK and DNA content. The validation parameters included specificity, recovery, linearity, precision, sensitivity, and stability. The lower limit of quantification was two CTCs per 8 ml peripheral blood. Samples were stable for 4 months in storage at -80°C. The applicability of the method was demonstrated by successful enumeration of CTCs, and the determination of DNA, and pERK before and after stimulation with EGF in 8 ml peripheral blood samples from patients with metastatic cancer.     

Two detergent-insoluble proteins of the human lymphocyte membrane are enriched in an isolated membrane fraction

Human lymphocytes isolated from peripheral blood on Ficoll/Paque density gradients were surface-labelled by ¹²⁵I/lactoperoxidase or ³H/reductive alkylation and lysed in buffer solutions containing non-ionic or amphoteric detergents (octylphenylpolyoxyethylenes, octylglucoside, cholylamidopropyldimethylammoniopropane sulfonate) under a variety of conditions. The cell lysate was fractionated by sedimentation or by density gradient centrifugation. The large majority of the labelled proteins is solubilized by the detergents. Two proteins of 45 000 and 30 000 molecular weight are the main detergent-insoluble, surface-labelled components. They can be fractionated from detergent lysates of cells in relatively pure form from the other membrane proteins and from nuclear material on density gradients. The same two proteins are specifically enriched in a membrane fraction isolated from a detergent-free cell homogenate by density gradient centrifugation. Cytoskeletal and other intracellular proteins remain associated with these two proteins when fractionated by either of these two independent methods.     

Properties of phase I purified cellular antigen of Coxiella burnetii

An attempt was made to purify phase I cell suspension of Coxiella burnetii used as an antigen in diagnostic serological tests. Homogenised suspension of chick embryos infected with phase I Henzerling and "Z" strains, after preliminary purification from host cell contaminants of chick embryos was subjected to consecutive centrifugation in sucrose/uropoline gradient and to continuous 20-45% uropoline gradient. The fractions obtained from uropoline gradient centrifugation were applied as phase I antigen C. burnetii in the following tests: complement fixation and microagglutination. Only fractions containing protein were serologically active. They proved to be of similar specificity and sensitivity as the antigens obtained by standard method. Moreover, it was found that after formalin treatment of C. burnetii cells no soluble antigens are liberated which could be detected by complement fixation test.     

Purification and properties of the Bombyx mori nuclear polyhedrosis virus liberated from polyhedra by dissolution with silkworm gut juice

Occluded virions of the Bombyx mori nuclear polyhedrosis virus were efficiently liberated from polyhedra by dissolution with the silkworm gut juice. The liberated virions were purified by sucrose density gradient centrifugation and the bands of enveloped virions were observed in the gradients. There was no functional difference between the gut juice-liberated and the carbonate-liberated virions. Disruption of enveloped virions by the gut juice was observed, but the formation of nucleocapsids from the degradation of the occluded virions was not detected. High yields of the enveloped virions from the polyhedra dissolved by the gut juice was obtained by separating the virions through sucrose density gradient centrifugation immediately after the dissolution of the polyhedra. Many factors, e.g., rearing seasons, silkworm strains, and rearing conditions, affect the polyhedra-dissolving property of the larval gut juice.