bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular ph in Dictyostelium: A 31P nuclear magnetic resonance study of its regulation and possible role in controlling cell differentiation
Authors: Kay R. R. , Gadian D. G. , Williams S. R.
Published: April 2021

Intracellular pH (pHi) has been measured in Dictyostelium discoideum cells by 31P nuclear magnetic resonance. Ax2 cells, newly harvested from growth medium, maintained a pHi of 7·33 ± 0·04 (17) at an extracellular pH ranging from 3·5 to 6·5. Below pH 3·5 the cells tend to lyse, whereas at pH values above 6’5 their pH; rises though they remain viable. pHi regulation in acid medium is not dependent on external Na+ or any other inorganic ion and so most probably involves the electrogenic plasma membrane proton pump.

No significant change in pHi was detected during development through to the slug stage. Mature stalk cells gave a very acidic phosphate signal (pH ⩽5·5) which was probably vacuolar in origin.

Indirect experiments had suggested that pHi might regulate the development of Dictyostelium cells, with low pH, favouring stalk cell and high pHi favouring spore cell differentiation. In particular, two inhibitors of the plasma membrane proton pump, diethylstilbestrol and zearalenone, had been shown to be stalk cell inducers. In the present studies measurements of pHi of cells exposed to these inducers failed to detect the expected drop in pHi. In addition, DIF-1 (a low Mr factor), the natural inducer of stalk cell formation, caused, if anything, a slight alka-linization of the cells. Thus the original theory linking pHi and cell differentiation is not supported by these results and therefore appears to require some modification.

Finally, extract experiments revealed the existence of two unidentified abundant phosphocompounds with resonant frequencies close to inorganic phosphate. The existence of these compounds can complicate the interpretation of spectra gained from living Dictyostelium cells.
The occurrence of lampbrush chromosomes in early diplotene oocytes of Xenopus laevis
Authors: Mitchell E. L. D. , Hill R. S.
Published: April 2021

Transmission electron microscopy has been used to study the chromosomes found in the early diplotene oocytes of Xenopus laevis. Chromosomes from 40 μm early diplotene oocytes were found to possess a normal lampbrush chromosome morphology. The contour length of the loops found on these chromosomes was measured, the mean value determined and compared with that for lampbrush chromosomes found on 300 μm mid-diplotene (Dumont stage II) oocytes. The mean contour length of the loops from 40 μm oocytes (12·33 ± 6·5 μm) was longer than that for the 300 μm oocytes (7·897 ± 5·22 μm). Analysis of variance showed these two values to be significantly different (p<0·01). The mean loop density (number of loops per 25 μm chromosome axis) was also determined for the two size classes of oocytes, being 9·33 ± 2·05 per 25 μm of chromosome axis for the 40 μm size class and 11·2 ± 2·435 per 25 μm of chromosome axis for the 300 μm size class. Analysis of variance showed these values to be significantly different (P< 0·01). The results of the present study demonstrate that the lampbrush chromosomes found in 40 μm early diplotene oocytes possess a greater loop contour length and a lower density of loops than those found in 300 μm, mid-diplotene oocytes. Possible reasons for these observations are discussed.
Kinetic properties of microtubule-activated 13 s and 21 s dynein ATPases: evidence for allosteric behaviour associated with the inner row and outer row dynein arms
Authors: Warner Fred D. , Mcilvain Jane H.
Published: April 2021

The 13 S and 21 S dynein ATPases from Tetrahymena cilia rebind to extracted doublet microtubules as inner row and outer row arms. Rebinding is accompanied by four- to ninefold activation of the ATPase activity. The soluble (microtubule-free) forms of the two dyneins exhibit simple saturation kinetics (h = 1·0) with Vmax ⪡ 1 μmol Pi mg-1 min-1 and Km= 20–40 μM-ATP. Mixing a fixed quantity of free dynein with increasing concentrations of extracted doublets results in systematic increases in all three kinetic parameters for each dynein. At infinite concentrations of doublets and ATP, each enzyme undergoes a significant shift to sigmoid saturation kinetics (h = 2–3), Vmax increases to a turnover rate of about 90mol ATP per mol Es-1 and the Michaelis constant increases to ⪢100 μM-ATP. These data suggest that both enzymes are allosteric and can be interpreted in terms of positive cooperativity relative to a minimum of two or three interacting sites. It is less clear whether this cooperativity is related to subunit interactions within the 21 S or 13 S particles, or to subunit interactions between adjacent particles (arms) on the microtubule lattice.
Structural integrity of the nuclear matrix: Differential effects of thiol agents and metal chelators
Authors: Dijkwel Peter A. , Wenink Paul W.
Published: April 2021

Nuclear matrices, associated with over 80% of the chromosomal DNA, could be isolated from BHK nuclei by extraction with 2M-NaCl. The matrices were found to impose at least two levels of structural order upon nuclear DNA.

From sedimentation studies it was inferred that metal depletion of the salt-extracted nuclei generated matrix structures, which sedimented at significantly lower rates than control matrices. Fluorescence microscopy revealed that the reduced sedimentation rate is a consequence of the increase in the radius of the DNA halo, i.e. the DNA loops emanating from the residual nucleus. Addition of Cu ions to nuclei prior to salt extraction was found to induce contraction of this DNA halo. These results indicate that Cu ions may play an important role in stabilizing one level of DNA folding.

When metal depletion had been brought about by thiol agents, a second effect was observed to occur. Within 15 min, salt-extracted nuclei disintegrated, generating irregularly shaped, slowly sedimenting structures. Disintegration only occurred when the full complement of DNA was still attached to the nuclear matrices.

Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that treatment with thiols did not detectably alter the polypeptide composition of DNA-depleted residual nuclei.

Results of these experiments suggest that both metal-protein interactions and disulphide bonds are important in maintaining higher-order structure in the nucleus. A model to account for these observations is discussed.
Nuclear division in the marine dinoflagellate Oxyrrhis marina
Authors: Xiao-Ping Gao , Jing-Yan Li
Published: April 2021

The nuclear division of Oxyrrhis marina is a very distinct one among the mitoses of dinoflagellates that have been studied. Using synchronized populations, we have investigated the ultrastructural changes in this nuclear division. In interphase, nuclei can be classified into two groups on the basis of the shapes of the chromosomes. Y- and U-shaped chromosomes have been observed in both types of interphase nuclei. By prophase the nucleus becomes oval, many nuclear plaques appear on the nuclear envelope, and many microtubules radiate from these nuclear plaques within the nucleus. Metaphase can be identified by the characteristic arrangement of the chromosomes; an equatorial metaphase plate is absent. As in many higher organisms, anaphase includes two stages: anaphase A and anaphase B. During anaphase A the nucleus does not apparently elongate and the chromosomes migrate towards the poles by a combination of the shortening of the chromosome-associated microtubules and the elongation of those located between daughter chromosomes. During anaphase B the nucleus elongates to about twice its former length. This elongation may result from growth of the interzonal nuclear envelope. Dividing nucleoli are associated with microtubules, which suggests that microtubules may play an active role in the division of the nucleolus.

The evolution of mitosis and the phylogenetic relationships between Oxyrrhis, typical dinoflagellates and Syndinium are discussed.
Cytochalasin-b-binding proteins related to glucose transport across the basolateral membrane of the intestinal epithelial cell
Authors: Uezato Tadayoshi , Fujita Michiya
Published: April 2021

Basolateral membrane vesicles were isolated from mouse enterocytes. The vesicles showed Na+-independent uptake of D-glucose. The uptake was inhibited by cytochalasin B and phloretin with 50% inhibition at 2’0 μM and 25 μM, respectively. 2-Deoxy-D-glucose inhibited D-glucose transport by 50% at 0·5 M. The basolateral membranes bound 13·5 p mol mg protein-1 of 0·1 μM-cytochalasin B. The effects of various monosaccharides on cytochalasin B binding were examined; the strongest inhibitor was 2-deoxy-D-glucose (20-30%). Sodium dodecyl sulphate-polyacryl-amide gel electrophoresis of the basolateral membranes labelled with [3H]cytochalasin B revealed two components with Mr values of 52(±2) and 30×103). Phloretin and 2-deoxy-D-glucose inhibited the photo-incorporation of [3H]cytochalasin B into these components. While phloretin inhibited the photolabelling of the two components to a similar extent, 2-deoxy-D-glucose seemed to inhibit preferentially that into the 52×103Mr component. The similar sensitivity to 2-deoxy-D-glucose of the photolabelling of the 52×103Mr component and of D-glucose transport, together with the fact that dithiothreitol removal increased the incorporation into the 52×103Mr component and decreased that into the 30×103 Mr component, seems to suggest that the 52×103Mr component is the major glucose transporter of the basolateral membrane.
Regulation of intracellular algae by various strains of the symbiotic Hydra viridissima
Authors: Bossert Patricia , Dunn Kenneth W.
Published: April 2021

In observations on three strains of green hydra, the host and the algal mitotic index is closely coordinated only for the smallest. As the hydra strain size increases the coordination of host and algal mitosis progressively breaks down, first in timing for a medium-sized strain and then in rate for a large strain. Despite disparities between host and algal mitotic index, the number of algae per host cell remains constant in all strains during the interval measured. To account for this constancy we suggest that the hydra may either prolong the duration of the algal tetraspore stage or cull excess algae.
Physical association between the peribacteroid membrane and lipopolysaccharide from the bacteroid outer membrane in Rhizobium-infected pea root nodule cells
Authors: Bradley Desmond J. , Butcher Geoffrey W. , Galfref Giovanni , Wood Elizabeth A. , Brewin Nicholas J.
Published: April 2021

Monoclonal antibodies were used as cytochemical markers to study surface interactions between endosymbiotic Rhizobium bacteroids from pea root nodules and the encircling peribacteroid membranes, which are of plant origin. Monoclonal antibodies that react with Rhizobium lipopolysaccharide (LPS) or with a plant membrane glycoprotein were used as markers for material from the bacteroid outer membrane or the peribacteroid membrane, respectively.

Membrane-enclosed bacteroids were isolated from nodule homogenates by sucrose gradient centrifugation, and the encircling peribacteroid membrane was released by mild osmotic shock treatment. Using an immunochemical technique (sandwich ELISA), it was shown that 1–5 % of the LPS antigen released into the peribacteroid fraction by mild osmotic shock treatment was physically associated with peribacteroid membrane through a detergent-sensitive linkage. This association could be visualized when freshly prepared peribacteroid material was immobilized on gold grids and examined by electron microscopy after dual antibody immunogold treatment and subsequent negative staining. The distribution of LPS antigen within infected nodule cells was also investigated by immunogold staining for thin sections of nodule tissue fixed in glutaraldehyde, and a close association between LPS antigen and peribacteroid membrane was often seen.
Nucleoside diphosphokinase, an enzyme with step changes in activity during the cell cycle of the fission yeast Schizosaccharomyces pombe: I persistence of steps after a block to the DNA-division cycle
Authors: Creanor J. , Mitchison J. M.
Published: April 2021

In confirmation of earlier results, nucleoside diphosphokinase is shown to be a ‘step’ enzyme in Schizosaccharomyces pombe with a sharp doubling in activity at the beginning of the cell cycle. These doubling steps occur at the same time in the cycle in the smaller cells of the mutant weel.6. An important result is that the activity steps persist with normal cell cycle timing after a block to the DNA-division cycle imposed by the cell cycle mutants cdc2.33 and cdc2.33 wee 1.6. This is clear proof that oscillatory controls of some cell cycle events can persist after the main periodic events of the DNA-division cycle have been abolished.
Assembly and remodelling of myofibrils and intercalated discs in cultured neonatal rat heart cells
Authors: Atherton B. T. , Meyer D. M. , Simpson D. G.
Published: April 2021

The reorganization of myofibrils and the re-formation of intercalated discs was examined in neonatal rat cardiac muscle cells during the first 72 h of culture. Rhodamine phalloidin was used to monitor the organizational state of the myofibrils and antibodies to desmoplakin and vinculin were used as markers for the presence of desmosomes and fasciae adherentes, respectively. Tiny punctate desmosomes were observed between muscle cells after 24 h and apparently increased in number and/or size between 24 and 48 h in culture. Fasciae adherentes were not detectable with antibodies to vinculin until after 48 h in culture. Well-defined sarcomeres were restored after 48 h in culture. Once formed the sarcomeric organization of the myofibrils was found to be stable provided they were attached to the sarcolemma via intercalated discs. However, if the myofibrils attached to regions of the membrane that lacked intercalated discs the sarcomeres appeared to break down gradually centripetally. When myofibrils attached to the membrane at the free edges of cells that were not in contact with other muscle cells, the striations stopped abruptly at a considerable distance before the myofibril attached to the membrane. These non-striated terminals elongated between 48 and 72 h and were associated with focal contacts that contained vinculin. Overall the results suggest that cell-cell contact may be critical for the stabilization of normal myofibrillar structure in the heart.
Isolation and culture of high endothelial cells from rat lymph nodes*
Authors: Ager Ann
Published: April 2021

The isolation and culture of an enriched population of cells from rat lymph nodes that have several properties of high endothelial cells are described.

High endothelial cells synthesize a unique sulphated glycolipid. This macromolecule in high endothelial cells was labelled with 35SO4 prior to cell isolation and was used to identify high endothelial cells after isolation. Collagenase digestion of pre-labelled lymph nodes yielded primary lymph node cultures in which two different cell types accounted for >90% of non-lymphoid cells isolated. The majority (>70%) were 20–30 μm diameter, round and 35S-labelled and were therefore high endothelial cells. The remaining unlabelled cells were 10–15 μm diameter and were identified as macrophages by phase-contrast microscopy. Isolated cells proliferated after 1–2 days and cultures were enriched for high endothelial cells as macrophages did not persist beyond 7–10 days. Small clumps (2–3 cells) of microvascular endothelial cells and/or adventitial fibroblasts were occasionally seen in primary cultures (⊁1% of isolated cells) but neither cell type proliferated. The identity of high endothelial cells was further substantiated using a polyclonal antiserum raised against lymph node cultures, which stained high endothelium in cryostat sections of lymph nodes. At confluence primary lymph node cultures bound lymphocytes as efficiently as high endothelium in lymphoid tissue and 40-fold more efficiently than rat aortic endothelial cells.

It is concluded that lymph node cultures contain high endothelial cells and that these cells continue to express surface determinants for lymphocytes in vitro.
Different mechanisms mediate structural changes and intracellular enzyme efflux following damage to skeletal muscle
Authors: Duncan C. J. , Jackson M. J.
Published: April 2021

Cellular damage, induced by either A23187 (2×10−5M) or 2,4-dinitrophenol (DNP) (10−3M), has been studied in incubated mouse soleus muscle and has been monitored by electron microscopy and measurement of creatine kinase (CK) efflux. CK efflux induced by DNP was dependent on extracellular Ca, whereas the characteristic myofllament breakdown was not. Chlorpromazine (2 ×10−4M; an inhibitor of phospholipase A (PLA2)) or 5 ×10−6M-nordi-hydroguaiaretic acid (NDGA) (a lipoxygenase inhibitor) almost completely inhibited CK efflux, following treatment with either A23187 or DNP, but did not affect myofilament damage. It is concluded that there are at least two separate pathways in cellular damage: (1) PLA2 activation and lipoxygenase activity culminating in sarco-lemma damage and (2) a system that produces characteristic destruction of the myofilament apparatus.
The ultrastructural localization of luciferase in three bioluminescent dinoflagellates, two species of Pyrocystis, and Noctiluca, using antiluciferase and immunogold labelling
Authors: Nicolas Marie-Thérèse , Sweeney Beatrice M. , Hastings J. Woodland
Published: April 2021

In order to discover the intracellular location of luciferase in dinoflagellates, sections from a number of species were treated with a polyclonal anti-luciferase and the bound antibody was visualized at the electron-microscope level by indirect immunogold labelling.

In two species of Pyrocystis and in Noctiluca, as in Gonyaulax, antibody became bound to dense vesicles, which correspond in size and position to light-emitting bodies detected in previous work. These vesicles resemble microsomes, are bounded by a single membrane and sometimes project into the vacuole.

Unexpectedly, the trichocysts of Gonyaulaxand Noctiluca and the related mucocysts of Pyrocystis also bound the antibody. This cross-reaction seems quite independent of bioluminescence, since the trichocysts of the non-luminous Cachonina also reacted positively. The possibility is discussed that a protein, different from luciferase but having some antigenic similarity, is present in trichocysts and related organelles.
Reactivation of cytoplasmic actomyosin in Physarum plasmodia extracted with glycerol and dimethylsulphoxide
Authors: Bell Renate , Achenbach Friedhelm
Published: April 2021

Thin-spread plasmodia of Physarum were subjected to extraction procedures using 50% glycerol or DMSO (dimethylsulphoxide) followed by labelling of actin with fluorescent phallotoxins.

During the reactivation of the actomyosin system by 2 mM-MgATP fluorescent actin fibres contract isotonically, which results in numerous fluorescent‘contraction beads’.

After short-term extraction 1 mM-Ca2+ has an inhibitory effect on the reactivation. This calcium sensitivity is abolished after long-term extraction with glycerol.

Calcium at 10 mM irreversibly inhibits reactivation, irrespective of the duration of extraction. The inhibitory effect of 10mM-calcium is prevented by phallotoxin labelling prior to incubation in Ca2+.

The DMSO model shows an improvement in structural preservation when compared with the glycerol models. However, reactivation is inhibited by prolonged treatment with DMSO.
Identification of protein antigens associated with the nuclear matrix and with clusters of interchromatin granules in both interphase and mitotic cells
Authors: Turner B. M. , Franchi L.
Published: April 2021

Monoclonal antibody 3C5 recognizes a family of protein antigens present predominantly within the nucleus of interphase cells. We have shown previously that the epitope recognized by 3C5 is phosphorylated and have concluded that the proteins defined by this antibody share a common phosphorylation site. Using a combination of immunofluorescence microscopy and immune-gold labelling in conjunction with electron microscopy, we have studied the distribution of 3C5-reactive material within interphase and mitotic cells. Antibody 3C5 was found to label specific structures within the interphase nucleus that, on the basis of their characteristic granulo-fibrillar morphology and strong staining with bismuth, have been identified as clusters of interchromatin granules (IG clusters). Double-labelling experiments with 3C5 and monoclonal antibodies to DNA have shown that these structures contain no detectable DNA. However, by indirect immunofluorescence, we have shown that 3C5-reactive nuclear structures do label with human autoantibodies to the Sm antigen, a component of small nuclear RNP particles (snRNPs). Granulofibrillar structures that stained strongly with bismuth, and were morphologically identical to nuclear IG clusters, were observed in the cytoplasm of mitotic cells. These structures also labelled with 3C5 but not with anti-Sm anti-bodies. Our results suggest that IG clusters remain essentially intact through mitosis though some snRNP components are apparently lost. In situ extraction of cultured cells with Triton X-100, micrococcal nuclease and 1–2M-NaCl failed to deplete 3C5-reactive material in either interphase or mitotic cells, though some redistribution was evident. In addition, 3C5-reactive proteins were identified in nuclear matrices prepared from rat liver by high-salt extraction procedures. However, the recovery of such proteins was strongly influenced by the preparation technique employed. Our results suggest that 3C5-reactive proteins and IG clusters are anchored to, but not integral components of, salt-resistant structural elements of the interphase nucleus and the mitotic cytoplasm, presumably the nuclear matrix and the cytoskeleton, respectively.
The origin and composition of nucleolus-like inclusions in the cytoplasm of fern egg cells
Authors: Bell P. R. , Pennell R. I.
Published: April 2021

Investigations of oogenesis in a number of lepto-sporangiate ferns have revealed nucleolus-like bodies in the cytoplasm of the maturing egg cells. In Pteridium and Lygodium these bodies appear to be aggregates of material secreted from protrusions of the nucleus. They are acidic in reaction, but autoradiography following feeding with [3H]uridine and the use of an RNase-gold probe, has failed to demonstrate the presence of RNA. Itis concluded that the bodies consist of acidic protein. Since their appearance coincides with an amoeboid condition of the nucleus and extreme dispersal of the chromatin, it is suggested that they represent a temporary storage in the cytoplasm of non-histone structural proteins of nuclear origin.
Conditional absence of mitosis-specific antigens in a temperature-sensitive embryonic-arrest mutant of Caenorhabditis elegans
Authors: Hecht R. M. , Berg-Zabelshansky M. , Rao P. N. , Davis F. M.
Published: April 2021

A monoclonal antibody, specific to phosphor-proteins in mitotic HeLa cells was found to cross-react with a similar set of proteins in embryos of the nematode, Caenorhabditis elegans. In C. elegans, as in mammalian cells, the highly conserved antigenic epitope is associated with a family of high molecular weight polypeptides. The antigenic reactivity of these multiple proteins also depends on their phosphorylation, since antibody binding is reduced after alkaline phosphatase treatment. The antigens are detected at the centrosomes, and in the nuclear region and surrounding cytoplasm of mitotic cells. The significance of these antigens is emphasized by their absence at restrictive temperature in embryos of the temperature-sensitive embryonic-arrest mutant, emb-29V. Furthermore, temperature shift-down experiments suggest that the emb-29 mutation defines a cell division cycle function that affects an essential activity required for progression into M phase.
Regulation of epitectin production in a malignant cell line
Authors: Bader Scott A. , Harris Henry
Published: April 2021

RT112 cells, a line derived from a human bladder carcinoma, produce epitectin at very low levels in standard culture media; but production and secretion of this mucin are greatly increased when the cells are exposed to hyperosmotic conditions. It appears that hyperosmolarity, by inducing an increase in intracellular sodium, entrains an increase in intracellular free calcium. Evidence is presented for the view that it is the increase in intracellular free calcium that provides the more direct stimulus for the enhanced production of epitectin.
Cytoplasmic ph at the onset of development in Dictyostelium
Authors: Aerts Rob J. , Durston Antony J. , Konijn Theo M.
Published: April 2021

Cytoplasmic pH (pH1) was measured by a pH ‘null point’ method in synchronous populations of Dictyostelium discoideum cells. Synchronous populations of vegetative cells with a lower pH1 than asynchronous populations of vegetative cells show a clear preference for the prespore differentiation pathway when they enter development. Treatment of these cells before development with the weak base ammonia results in an increase in pH1 and an abolition of the preference of the cells for the prespore pathway. On the other hand, synchronous populations of vegetative cells with the same pH) as asynchronous populations of vegetative cells resemble these last cells in that they show no preference for a certain differentiation direction when they enter development. However, both these synchronous and asynchronous populations of cells do acquire a preference for the prespore pathway when they are treated before development with the weak acid 5,5-dimethyl-2,4-oxazolidineolione (DMO), which lowers their pH). It is concluded that changes in pH1 at the onset of the developmental program lead to significant changes in the preference of the cells for the prespore differentiation pathway in Dictyostelium discoideum.
Synaptonemal complex spreading in Allium ursinunr. pericentric asynapsis and axial thickenings
Authors: Loidl Josef
Published: April 2021

In Allium ursinum meiotic pairing of homologues is always incomplete; a proximal region on each bivalent remains regularly unsynapsed even in late pachytene. The spatial correlation of the unsynapsed region with the kinetochore suggests that the kinetochore itself exerts an inhibitory effect on synapsis in its vicinity. This can be interpreted as the cytological basis of the ‘centromere effect’ on recombination in this species. Moreover, the high incidence of a pericentric inversion loop in a heterozygous chromosome pair shows that proximal pairing initiation is possible and that its failure cannot be responsible for pericentric asynapsis.

The formation of the inversion loop is complicated by the need for two independent pairing initiation sites because synapsis cannot proceed across the pericentric region. It is proposed that the meiotic bouquet polarization helps in establishing the presynaptic alignment of the homologous sites within the inverted regions and hence to achieve a high rate of inversion loop formation.

Thickenings of the axial/lateral elements are not distributed equally along the synaptonemal complex. They are underrepresented in unpaired axes but strikingly abundant at the borders with synapsed regions, suggesting their origin in the pairing forks during the process of synapsis. They are virtually always present at nucleolusorganizing regions and often they appear at corresponding sites on opposite lateral elements. Besides the thickenings several other kinds of axial deformities are present in unpaired axes.
Reticuloplasmins: a novel group of proteins in the endoplasmic reticulum
Authors: Koch G. L. E.
Published: April 2021

The existence of a special milieu within the endoplasmic reticulum (ER) has been well known to microscopists because of its special osmiphilic properties and the term ‘reticuloplasm’ was coined to describe the contents of the ER (Krstic, 1979). The purpose of this article is to bring together some recent observations showing that reticuloplasm does indeed contain a very special complement of its own resident proteins, referred to as reticuloplasmins, and to consider the possible role of these proteins in ER structure and function.
Enhancement Of Pheomelanogenesis By L-Dopa In The Mouse Melanocyte Cell Line, Tm10, In Vitro
Authors: Sato Chikara , Ito Shousuke , Takeuchi Takuji
Published: April 2021

Cells of TM10, an established cell line, are melanocytes that contain equal amounts of eumelanin (black pigment) and pheomelanin (yellow pigment). The content of pheomelanin drastically increased when the cells were cultured in growth medium containing 0·2mM-L-dopa (L-dihydroxyphenylalanine), which is the common precursor for both eumelanogenesis and pheomelanogenesis.

After this treatment, the amount of pheomelanin was 3·7-fold greater than that of control in TM10, whereas the amount of eumelanin changed very little. In contrast, 5-S-cysteinyl-dopa, which is the specific precursor for pheomelanogenesis downstream of L-dopa, did not cause preferential increase in pheomelanogenesis. Ultrastructural observations also confirmed these results; in 0·2 mM-L-dopa, an increase in the number of pheomelanosomes was observed in the cytoplasm of TM10 cells. Our results also suggest that the L-dopa treatment results in a decrease in tyrosinase activity per melanosome.
Biochemical characterization of isolated CHO cell mitotic spindles: identification of calmodulin-binding proteins
Authors: Brady Richard C. , Schibler Matthew J. , Dedman John R. , Cabral Fernando
Published: April 2021

Mitotic spindles were isolated from Chinese hamster ovary (CHO) cells and examined morphologically and biochemically. The isolated spindles were observed to be intact structures containing associated chromosomes and were surrounded by a cage of vimentin-containing filaments. Two-dimensional gel electrophoresis of isolated spindles versus whole cell homogenates indicated that isolated spindles were free from significant cytoplasmic contamination and contained tubulin, actin, vimentin and an 80×103Mr quadrapeptide as their major protein constituents. Five calmodulin-binding proteins with molecular weights of 200, 160, 130, 60 and 52(×103) Mr were identified within isolated spindles. These calmodulin-binding proteins may be involved in regulating microtubule organization and depolymerization during karyokinesis.
Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F
Authors: Owen M. E. , Cavé J. , Joyner C. J.
Published: April 2021

Fibroblastic colonies, each of which is derived from a single precursor cell (CFU-F), are formed when suspensions of marrow cells are cultured in vitro. The ability of marrow CFU-F to differentiate in vitro was investigated using the expression of alkaline phosphatase activity as a marker for osteogenic differentiation. In cultures of rabbit marrow cells the colonies formed varied in size, morphology and expression of enzyme activity, indicating that marrow stromal CFU-F are a heterogeneous population. Growth and differentiation of marrow CFU-F can be modified in vitro. Epidermal growth factor increased average colony size and reduced clonal expression of alkaline phosphatase activity to very low levels. Hydrocortisone activated the osteogenic differentiation programme within the cellular progeny of a wide spectrum of CFU-F. The results support the possible development of in vitro clonal methods for the study of differentiation and regulation of the osteogenic and other fibroblastic cell lines of the marrow stromal system.
The rearrangement of cytoskeletal systems in epithelial cells accompanying the transition from a stationary to a motile state at the start of epithelial spreading
Authors: Takeuchi Shigeo
Published: April 2021

The changes in the alignment of F-actin bundles in epithelial cells were studied, mainly using NBD-phallacidin, as the isolated corneal epithelium started to spread along the surface of Millipore filter.

The epithelial cells at the margin were found to make the transition from a stationary to a motile state in two steps: first, from polygonal cells, in which F-actin was deposited along the cell border, to hemispherical cells, in which F-actin was distributed homogeneously along the inner cell surface; and second, from the hemispherical cells to spread cells in which F-actin bundles ran radially or in parallel to each other through the leading lamella from a cytocentre to the edges, as in fibroblastic cells. During the second step, an amorphous mass of F-actin was transiently accumulated at the distal end in the half-extended lamella and structured into bundles during development of the lamella. Since this proceeded only on substrata to which marginal cells could make contact firmly enough to enable the cells to extend, it was concluded that F-actin bundles were aligned within extending lamellae in relation to the tension exerted by the lamellae, and might be associated with other components to be microfilaments.
How are invertebrate epithelia made tight?
Authors: Skaer H. le B. , Maddrell S. H. P.
Published: April 2021

Many compartments in the animal body are delineated and separated by epithelial sheets of cells. Such epithelia are described as leaky or tight depending on the extent to which the compartments on either side of them appear to be kept distinct. It has been assumed that a‘tight’ epithelium has intercellular junctions that completely seal the paracellular pathway. Indeed, in vertebrates, some tight junctions have been shown to be virtually impenetrable. However, in invertebrate tight epithelia, there is controversy over whether the paracellular pathway is sealed by a vertebrate type of tight junction or whether the septate junction, a special invertebrate junction not seen in vertebrate tissues, can fulfil the same function.
Refractile bodies (R bodies) from the macronuclear killer particle Caedibacter caryophila
Authors: Schmidt Helmut J. , Pond Finn R. , Görtz Hans-Dieter
Published: April 2021

The macronuclei of certain isolates of Paramecium caudatum are inhabited by the bacterial endonucleobiont Caedibacter caryophila. These symbionts confer upon host paramecia a killer trait, which is clearly associated with the presence of R bodies in the endosymbionts.

R bodies are unique inclusion bodies of caedi-bacteria (which are obligate endosymbionts of Paramecium) and of certain free-living bacteria of the genus Pseudomonas. They have been grouped into four classes on the basis of morphology and behaviour. A fifth R body type, introduced in this study, is named the Cc R body to indicate its presence in bright particles of Caedibacter caryophila. Type Cc R bodies are approximately 0–8 pm in width and diameter. They unroll in a telescopic fashion from the inside and are distinguished by a tapered inner and a blunt outer terminus. They are further associated with icosahedral bacteriophages, which stick to the inner terminus. Cc R body proteins show a ladder-like pattern of stained bands on polyacrylamide gels and exhibit a certain degree of antigenic cross-reactivity with type 51 R bodies.
Interaction of plasma membrane-associated filaments and H-2 histocompatibility antigens before and after induced patching and capping
Authors: Feltkamp Constance A. , Spiele Hermina , Roos Ed
Published: April 2021

The interaction of H-2 antigens and plasma membrane-associated filaments was studied on dry-cleaved preparations of immunogold-labelled lymphoma cells. In prefixed cells, the plasma membrane-associated network was isotropic without any prevailing direction of the filaments, and the gold-labelled H-2 antigens were preferentially localized over or at a very short distance from membrane-associated filaments. Incubation of unfixed cells with anti-H-2 antibodies followed by fixation and incubation with anti-Ig, did not induce detectable redistribution of H-2 antigens or of the filament network. Notwithstanding this apparent absence of rearrangement of H-2 antigens and filaments, a detergent-resistant linkage to the cytoskeleton was induced. Before immune incubations, virtually all H-2 antigens were solu-bilized by extraction with Triton X-100, while after incubation with anti-H-2 antibodies about 50 % of the H-2 antigens were linked to the Triton X-100-insoluble cytoskeleton. Sequential addition of anti-H-2 and anti-Ig antibodies to unfixed cells induced formation of patches and caps of H-2 antigens. Under these conditions, the majority of the H-2 antigens became linked to the detergent-resistant cytoskeleton. Redistribution into patches and caps was often accompanied by a local rearrangement of the isotropic network into bundles of parallel filaments immediately adjacent to the plasma membrane. Patches were seen to overly both isotropic networks and these parallel filaments. Large sheets of plasma membrane overlying parallel filaments were frequently devoid of gold-labelled H-2 antigens and coated pits, and thus most probably represented areas away from caps. This observation suggests that capping is accompanied by a rearrangement of filaments close to the membrane.
Sex-chromosome anaphase movements in crane-fly spermatocytes are coordinated: Ultraviolet microbeam irradiation of one kinetochore of one sex chromosome blocks the movements of both sex chromosomes
Authors: Swedak Julia A. M. , Forer Arthur
Published: April 2021

Sex chromosomes in crane-fly spermatocytes move polewards at anaphase after the autosomes have reached the poles. In Nephrotoma abbreviate the sex chromosomes are 8 μm long by 3·5 μm wide and have two orientations when they move: the long axis of the sex chromosome is either perpendicular or parallel to the spindle axis. We assume (1) that when a sex chromosome is perpendicular to the spindle axis it has a chromosomal spindle fibre to each pole, one from each kinetochore, as in other species; and (2) that when a sex chromosome is parallel to the spindle axis each kinetochore has spindle fibres to both poles, i.e. that the latter sex chromosomes are maloriented.

We irradiated one kinetochore of one sex chromosome using an ultraviolet microbeam. When both sex chromosomes were normally oriented, irradiation of a single kinetochore permanently blocked movement of both sex chromosomes. Irradiation of non-kinetochore chromosomal regions or of spindle fibres did not block movement, or blocked movement only temporarily. We argue that ultraviolet irradiation of one kinetochore blocks movement of both sex chromosomes because of effects on a ‘signal’ system. The results were different when one sex chromosome was maloriented. Irradiation of one kinetochore of a maloriented sex chromosome did not block motion of either sex chromosome. On the other hand, irradiation of one kinetochore of a normally oriented sex chromosome permanently blocked motion of both that sex chromosome and the maloriented sex chromosome. We argue that for the signal system to allow the sex chromosomes to move to the pole each sex chromosome must have one spindle fibre to each pole.
Mapping cell–glass contacts of Dictyostelium amoebae by total internal reflection aqueous fluorescence overcomes a basic ambiguity of interference reflection microscopy
Authors: Todd I. , Mellor J. S. , Gingell D.
Published: April 2021

The widespread ability of eukaryotic cells to produce thin cytoplasmic sheets or lamellae 100–200 nm thick can give rise to uncertainties in the interpretation of interference reflection microscopy (IRM) images when cell–substratum topography is the key interest. If allowed to spread upon a poly-L-lysine-coated surface, Dictyostelium discoideum amoebae typically form ultrathin lamellae of ≈100nm thickness by cytoplasmic retraction. Whereas the cell body is grey, the lamellae appear very dark under IRM optics. These dark areas could be misinterpreted as stemming from a closer cell–substratum apposition beneath the lamellae than the cell body. This ambiguity can be avoided if the technique of total internal reflection aqueous fluorescence (TIRAF) is used in conjunction with a high refractive index glass (n = 1·83) as substratum. Contributions to the image generated by thin cytoplasm and also variable cytoplasmic refractive index are thereby minimized due to the extremely short range of the ‘illuminating’ evanescent wave. From our comparative IRM and TIRAF study of the ultrathin lamellae of Dictyostelium amoebae it is concluded that the cell–glass gap is relatively uniform beneath the entire cell. We briefly discuss the sensitivity of several cell types to TIRAF, the generation of ultrathin lamellae and the nature of the cell–glass gap.
Localization of platelet antigens and fibrinogen on osteoclasts
Authors: Athanasou N. A. , Quinn J. , Heryet A. , O’D. Mcgee J.
Published: April 2021

The antigenic phenotype of the human osteoclast, which is known to be derived from a circulating mononuclear precursor cell of haemopoietic origin, is controversial. Recent studies have shown that macrophage as well as megakaryocyte/platelet antigens are expressed by osteoclasts. In this study, we have sought to define, by immunohistochemistry, the nature and possible function of platelet antigens expressed by human osteoclasts in foetal and adult bone specimens. Monoclonal antibodies to platelet glycoprotein IIIa (gpllla) and CD9 antibodies stained osteoclasts in all bone specimens examined. Fibrinogen was also localized to the osteoclast membrane in foetal bone imprints. In addition, we found that CD9 and gpllla antibodies reacted weakly with monocytes in buffy coat smears. Antibodies to factor 8 and glycoproteins lb and IIb/IIIa did not react with osteoclasts. These results show that osteoclasts, monocytes, macrophages, megakaryocytes and platelets possess common antigens and that fibrinogen is present on the surface of osteoclasts. By analogy with platelets, CD9 and gpllla may play a role in fibrinogen binding by osteoclasts. Possible mechanisms by which platelet antigens and fibrinogen binding could affect osteoclast function are proposed.
Distribution of pericentriolar material in multipolar spindles induced by colcemid treatment in chinese hamster ovary cells
Authors: Sellitto Caterina , Kuriyama Ryoko
Published: April 2021

Mitotic Chinese hamster ovary cells were obtained by treatment with microtubule drugs under various conditions, and the shape of spindles was analysed by phase-contrast microscopy of isolated spindles, and by indirect immunofluorescence staining of whole mitotic cells with anti-tubulin antibody. Bipolarity of spindles was maintained after treatment with 0·05 μM of colcemid for 3·5 h, but increased exposure to higher concentrations (0·32 μM) and for longer durations (5·5 h) led to a marked rise in multipolar spindles. Nocodazole treatment, on the other hand, failed to show a multiplicity of spindle poles even at 3·3 μM. Each pole of a multipolar spindle was associated with pericentriolar material, as shown by staining with an autoimmune serum specific for pericentriolar material. The number of locations with free pericentriolar material capable of polymerizing microtubules in vitro also increased with increasing numbers of spindle poles, suggesting that dispersion of the pericentriolar material resulted in the production of many microtubule-nucleating sites in multipolar spindles. The different efficiencies of recovery from different drugs, which have been known to be quite variable, may be partly due to the different extent of dispersion of the pericentriolar material.
Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes in culture
Authors: Watt Fiona M.
Published: April 2021

Articular chondrocytes are known to be pheno-typically unstable in culture. One condition that has been reported to suppress dedifferentiation is cultivation at high density on tissue-culture plasΛtic. The aim of the experiments described here was to study the effect of seeding density on chondrocyte proliferation and 35SO4 incorporΛation, and on the types of collagen and proteoglyΛcan synthesized. I found that cells seeded at low or high density reached the same final density at confluence, and that 35SO4 incorporation, while initially higher (per cell) in high-density cultures, fell under both conditions, reaching the same low level after 3 weeks. The proportion of cells expressing keratan sulphate fell in low-but not high-density cultures and the decline was not prevented by inhibition of cell division. In all the cultures cells expressing keratan sulphate tended to have a rounded morphology. After 21 days inculture, chondrocytes grown at high density exΛpressed predominantly large proteoglycans that aggregated with hyaluronic acid, whereas in low-density cultures a smaller, non-aggregating form was also present. By 21 days in culture cells at both high and low density were expressing type I collagen, although the high-density cells also had an extensive extracellular matrix of type II colΛlagen. These observations support the conclusion that high seeding density stabilizes the chondroΛcyte phenotype to a greater extent than low seedΛing density. They also suggest that enhanced dedifferentiation at low density may be due to cell spreading, rather than to selective proliferation of a phenotypically unstable subpopulation of cells.
The effect of co2 on the timing of cell cycle events in fission yeast Schizosaccharomyces pombe
Authors: Novák B. , Halbauer J. , László E.
Published: April 2021

The effect of CO2 removal on the cell cycle phases of Schizosaccharomyces pombe has been examined in minimal, aspartate-containing and complete medium. The removal of CO2 shortened the G2 phase of the cell cycle and arrested the cells in G1 phase in minimal medium. The Gi block caused by C02 deprivation was demonstrated by transition-point and flow-cytometry analyses. The slow-down of anapleurotic CO2 fixation might be responsible for this effect, as aspartic acid could abolish the G1 block. The shortening of G2 phase in the wild-type cells was observed in every medium irrespective of whether the growth rate was changed or not. The experiments in which growth rate was not changed by CO2 shift-down suggest that this CO2 effect can be independent from its action on CO2-fixing steps in metabolism.

Therefore we propose that CO2 inhibits mitosis in fission yeast and we explain the proportionality between growth rate and cell size at mitosis found by Fantes & Nurse by this CO2 inhibition. The larger CO2 production in fast-growing cells leads to a higher CO2 concentration, which could exert a stronger inhibition of mitosis. A wee mutant, which has lost its mitotic size control, also shows the G1 block after CO2 deprivation, but its mitosis is insensitive to CO2. Comparing the respiration of wee and wild-type cells we conclude that CO2 inhibits the citric acid cycle in the wild type. The consequence of these results in the regulation of fission yeast cell cycle is discussed.