bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular electrical potential measurements in Drosophila follicles
Authors: Bohrmann Johannes , Huebner Erwin , Sander Klaus , Gutzeit Herwig
Published: April 2021

We measured the intracellular electrical potential in oocyte and nurse cells of Drosophila follicles at different developmental stages (6–14) and determined the intrafollicular potential difference. During stages 8—10B, when intrafollicular transport is known to occur, no significant potential difference was found. During late vitellogenic stages the nurse cells assume a more positive potential than the oocyte. This result contrasts with the published data on Hyalophora follicles, in which intercellular electrophoresis of negatively charged proteins occurs from nurse cells to oocyte as a result of an intrafollicular potential difference (nurse cells more negative than the oocyte). Such a potential difference was not observed in Drosophila follicles at any stage, not even after application of juvenile hormone. The extrafollicular electrical field is described with a dipole model. The hypothetical dipole is located in the long axis of the follicle and changes its calculated length stage-specifically.
Anti-tubulin antibodies locate the blepharoplast during spermatogenesis in the fern Platyzoma microphyllum R.BR.: A correlated immunofluorescence and electron-microscopic study
Authors: Doonan John H. , Lloyd Clive W. , Duckett Jeffrey G.
Published: April 2021

The discovery that the monoclonal anti-tubulin antibody YOL 1/34 recognizes a microtubule organizing centre, the BIepharoplast (which arises de novo during the latter stages of spermatogenesis in the fern, Platyzoma microphyHum), has enabled us to follow it and associated microtubules throughout most of its ontogeny. By correlating electron-microscopic and immunofluorescence observations, YOL 1/34 is seen to stain the BIepharoplast uniformly at a time when no microtubules are present within the organelle. Later, staining becomes intense at the surface, concomitant with the re-location of cylindrical channels to the periphery of the BIepharoplast.

During anaphase of the ultimate division of the spermatid mother cell the BIepharoplast moves to the spindle poles and sharpens the otherwise barrel-shaped mitotic apparatus. Prior to this stage the BIepharoplast is, however, off-centre and at variable positions around the poles.

Later still, in the differentiating spermatids, the BIepharoplast is the focus for radiating cytoplasmic microtubules that abut directly onto the electron-dense organelle, penetrating the ribosome-free halo.

The three main conclusions are: (1) that tubulin in a pre-microtubular form is associated with the cylindrical channels that arise de novo within the previously amorphous BIepharoplast and act as a template in basal body formation; (2) that the late appearance of the BIepharoplast as a focus for the spindle poles during the final mitosis provides strong argument against its functioning during spindle pole initiation (despite its ability to sharpen the poles at anaphase); (3) that the BIepharoplast does seem to act as a microtubule organizing centre in the mitotically quiescent spermatid.
Elemental concentration gradients between subcellular compartments
Authors: Cameron Ivan L. , Hansen John T. , Hunter Keithley E. , Padilla George M.
Published: April 2021

To gain information on the mechanisms involved in the establishment and maintenance of subcellular gradients of Na, K, Cl and other elements in the flagellate, Euglena gracilis, we turned to the technique of ultracentrifugal stratification of its intracellular contents, which is achieved without loss of viability or cell rupture. Stratified and non-stratified Euglena were cryofixed for energy-dispersive X-ray microanalysis of Na, K, Cl and other elements in thin freeze-dried cryosections. A number of significant elemental concentration differences (expressed as mmol kg−1 dry weight) were found between chloroplast, nucleus, paramylon granules and open cytoplasm (which contained ribosomes, membranes and macromolecules associated with the cytomatrix) in the non-stratified cells. Stratification caused several ions to be redistributed. For example, we observed a significant increase in K and Cl in the nucleus, which was correlated with the condensation of chromatin. Also Cl, but not Na, decreased significantly in the region of cytoplasm that was cleared of observable ribosomes, membranes and macromolecules associated with the cytomatrix, as well as of observable cytochemical enzyme activity. We conclude from the data that more than half of the Cl in open cytoplasm was adsorbed to or entrapped in material that was removed by ultracentrifugation. Thus, it appears that a close association of at least one ion, Cl, with ultracentrifugable material is involved in maintenance of the measured Cl concentration in the open cytoplasm of the non-stratified cell.
Actin in plants
Authors: Lloyd Clive
Published: April 2021

It has been known for some time that actin exists in lower plants, the most familiar examples being provided by certain algae in which actin constitutes the cytoplasmic cables along which particles and organelles stream. The choice of material was an important part of determining the identity of the filaments because these giant filamentous cells were capable of being broken open to expose cytoplasmic strands, which could then be decorated with heavy meromyosin (HMM) (reviewed by Williamson, 1986).
Cell adhesion and phagocytosis promoted by monoclonal antibodies not directed against fibronectin receptors
Authors: Grinnell Frederick , Ho Chin-Han , Tuan Tai-Lan
Published: April 2021

In this report we describe cell adhesion and phagocytosis promoted by two monoclonal antibodies that were selected for immunofluorescence staining of non-permeabilized baby hamster kidney (BHK) cells. Anti-BHKl staining was heaviest along cell margins, whereas anti-BHK2 staining was continuous along cell margins. Neither antibody stained elongated plaque structures such as were observed when cells were reacted with antibodies to fibronectin (FN) receptors. The monoclonal antibodies functioned as adhesion ligands in four different assays: attachment to culture dishes, spreading, binding of latex beads and phagocytosis. Anti-BHKl and anti-BHK2 promoted attachment to culture dishes similarly, but anti-BHK2 was more effective at promoting cell spreading. Antibody-promoted cell spreading was inhibited by the peptides Ser-Asp-Gly-Arg and Gly-Arg-Gly-Asp-Ser-Pro but not by other, related, peptides tested. The monoclonal antibodies also promoted binding of latex beads, and the bead binding sites were motile, on the basis of their ‘capping’ response. Nevertheless, anti-BHK2 beads were phagocytosed by cells 5-to 20-fold more efficiently than anti-BHKl beads. The binding sites for anti-BHKl and anti-BHK2 were characterized by immunoprecipitation experiments. Anti-BHKl binding sites contained 50K (K= 103Mr) and 88K components under non-reducing conditions that migrated as a 51/53K doublet and a 93K component under reducing conditions. On the other hand, anti-BHK2 binding sites contained 88K and 110K components under non-reducing conditions that shifted to apparent 107K and 128K values when measured under reducing conditions.
Secretion of adhesive material during encystment of Phytophthora cinnamomi zoospores, characterized by immunogold labelling with monoclonal antibodies to components of peripheral vesicles
Authors: Gubler Frank , Hardham Adrienne R.
Published: April 2021

During encystment, zoospores of the dieback fungus Phytophthora cinnamomi attach themselves to the plant root surface by secretion of adhesive material. Previous workers have proposed, on the basis of ultrastructural studies, that the adhesive material is released by rapid exocytosis of large vesicles that underlie the zoospore plasma membrane. In the present study a monoclonal antibody (Cpa-2) that binds to the adhesive material has been used to investigate the sites of storage of this material in the zoospores, and the timing and pattern of its secretion during encystment. Immunolabelling of thin sections of zoospores with Cpa-2 followed by gold-labelled secondary antibodies has revealed that the adhesive material is not stored in the large peripheral vesicles but in a population of small vesicles in the cell periphery. In studies of other Oomycete fungi, these small vesicles have usually been described as microbodies. Another monoclonal antibody (Lpv-1) does bind to the contents of the large peripheral vesicles. Double immunolabelling of zoospores and encysting cells with Cpa-2-Au10 and Lpv-1-Aui8, coupled with morphometric analysis, has shown that 85% of the adhesive material is secreted within the first minute following induction of encystment and that the large peripheral vesicles are not exocytosed during encystment but instead tend to migrate towards the centre of the cell. Immunoblot analysis with Cpa-2 shows the presence of several high molecular weight bands (Mr> 300 000) which also react with the lectin, soybean agglutinin. These high molecular weight components are susceptible to digestion with Pronase. Lpv-1 also reacts with high molecular weight bands but of different mobility from those recognized by Cpa-2. Concanavalin A binds to bands corresponding to those labelled by Lpv-1. We conclude that adhesion of cysts to plant roots involves the secretion of high molecular weight glycoproteins by rapid exocytosis of small peripheral vesicles and that the material contained in the large peripheral vesicles is also glycoprotein in nature but is not secreted during encystment.
Introduction of a porcine growth hormone fusion gene into transgenic pigs promotes growth
Authors: Vize Peter D. , Michalska Anna E. , Ashman Rod , Lloyd B. , Stone B. A. , Quinn P. , Wells J. R. E. , Seamark R. F.
Published: April 2021

Six transgenic pigs have been produced by microinjecting a human metallothionein promoter/porcine growth hormone gene construct into the pronuclei of fertilized eggs which were transferred to synchronized recipient sows. The resulting transgenic animals contained between 0·5 and 15 copies of the gene construct per cell, and at least one of the animals expressed the introduced gene and grew at an increased rate compared to both transgenic and non-transgenic littermates. Some of the transgenic animals that did not appear to grow at increased rates were found to contain rearranged gene sequences. Two of the transgenic pigs have been shown to pass on the introduced genes to their offspring.
Spectrin-like proteins in the paraflagellar rod structure of Trypanosoma brucei
Authors: Schneider André , Lutz Hans U. , Marugg Richard , Gehr Peter , Seebeck Thomas
Published: April 2021

A polyclonal, monospecific rabbit antibody to human erythrocyte spectrins cross-reacted with two sets of proteins (a doublet of 180/200K and a triplet of 67–66–65K; K= 103Mr) in the parasitic protozoon Trypanosoma brucei brucei. Except for the 66K protein, the cross-reacting proteins are localized in the flagellum, on the basis of evidence from cell fractionation and immunofluorescence microscopy. Immunogold labelling and electron micrographs further revealed that the spectrin-like proteins are confined to the paraflagellar rod structure. The spectrin-like proteins with apparent molecular weights of 180 and 200 share homology with spectrin band 1, since V8-protease from Staphylococcus aureus generated similarly sized, antigenic peptides from these proteins. The results indicate homology between the cross-reacting proteins and human red cell spectrin.
A contractile cytoskeletal network ofParamecium:the infraciliary lattice
Authors: De Loubresse Nicole Garreau , Keryer Guy , Viguès Bernard , Belsson Janine
Published: April 2021

Among the various cytoskeletal networks and structures that compose the highly organized cortex of Paramecium, the infraciliary lattice (ICL) is the innermost one and marks the cortical boundary. It is made up of filamentous bundles running around the proximal ends of basal bodies. Immunofluorescence studies, using two different antibodies specifically labelling the network, allowed us to describe the disorganization-reorganization cycle of the ICL during division, and observations by immunoelectron microscopy provided new information on its ultrastructure. A partial purification of large fragments of a contracted form of the network was obtained. Among the proteins from ICL-enriched fractions, two 23 to 24(×103)Mr polypeptides were shown to cross-react with a polyclonal antibody raised against 22 to 23(×103)Mr Ca2+-binding proteins from the microfibrillar ectoplasmic-endoplasmic boundary of Isotricba and to be selectively solubilized in the presence of EGTA.

The possibility that the ICL may be a new example of a non-actin contractile system found in lower eukaryotes is discussed.
Formation of cell outgrowths by external force: A model study
Authors: Popov S. V. , Margolis L. B.
Published: April 2021

Cell outgrowth formation by a force applied to the external membrane was investigated. This extrinsic force, which is generated in a special chamber by an alternating current (AC) electric field (EF) of high frequency, pulls the membrane outwards. The force arises due to the redistribution of ions beneath the cell surface and is applied to the thin (10–20 Å) undermembrane layer. This force is sufficient to generate cell-specific membrane protrusions of various types. The morphology of these protrusions in mouse embryo and human fibroblasts, and in mouse L cells, resembles that of the outgrowths that these cells form under normal physiological conditions. Ehrlich ascites tumour cells (which normally grow in suspension) in an AC EF form only thin short processes. Erythrocytes when subjected to EF treatment are not capable of producing processes at all. An aligned system of microfilaments was found in the outgrowths produced by mouse embryo fibroblasts. However, cytochalasin B, sodium azide + 2-deoxy-D-glucose, carbonyl-m-chlorphenylhydrazon + 2-deoxy-D-glucose, colcemid and incubation at 4°C were not able to prevent the generation of processes in an EF. It is proposed that the morphology of cell protrusions is determined by the structure of the domains in the plasma membrane, rather than by active cytoskeletal reorganization. The experimental system that we developed provides an opportunity for studying the mechanical linkages of cytoskeletal elements with the plasma membrane.
Foetal and cancer patient fibroblasts produce an autocrine migrationstimulating factor not made by normal adult cells
Authors: Schor Seth L. , Schor Ana M. , Grey Ann Marie , Rushton Graham
Published: April 2021

We have previously reported that (1) the migration of foetal and adult fibroblasts into threedimensional collagen matrices is differentially affected by cell density, and (2) skin fibroblasts from cancer patients commonly display a foetal-like mode of migratory behaviour. Data presented here indicate that differences in the migration of these cell types are particularly apparent in cultures plated at high density (i.e. at cell confluence); under these conditions, foetal fibroblasts and the foetal-like fibroblasts of cancer patients migrate into the three-dimensional collagen matrix to a significantly greater extent than do normal adult cells. In this initial study concerned with the biochemical basis of these observations, we report that medium conditioned by either foetal or cancer patient fibroblasts stimulates the migration of confluent adult cells. This stimulation of migration is specific to confluent cells, as the migration of subconfluent adult fibroblasts is unaffected by these conditioned media. Gel filtration chromatography of foetal fibroblast-conditioned medium indicates that migration-stimulating activity is recovered in a single peak with an apparent molecular mass in the range of 50–60 (×103). The active migration stimulating factor (MSF) in both foetal and cancer patient fibroblast-conditioned media appears to be a protein stable at acid pH, but inactivated by heat, alkaline pH and reductive alkylation. MSF produced by foetal and cancer patient fibroblasts is presumably responsible for the characteristically elevated levels of migration displayed by these cells in confluent culture, thereby suggesting an autocrine mode of action for this factor. Stimulation of adult cell migration by MSF requires the presence of either serum or plateletpoor plasma and is not observed in serum-free medium. MSF does not appear to affect either the proliferation or morphology of normal adult cells under any of the culture conditions examined.
Fibroblasts from cancer patients display a mixture of both foetal and adult-like phenotypic characteristics
Authors: Schor Seth L. , Schor Ana M. , Rushton Graham
Published: April 2021

We have previously reported that foetal and adult fibroblasts display distinctive migratory phenotypes when cultured on three-dimensional collagen gels. Both skin and tumour-derived fibroblasts from a significant proportion of patients with breast cancer were subsequently observed to display foetal-like migratory behaviour. In the accompanying paper concerned with the biochemical basis of these observations, we presented evidence that foetal fibroblasts and the foetal-like fibroblasts of cancer patients produce a soluble migration-stimulating factor (MSF) not made by normal adult cells. Data are presented here indicating that: (1) the spontaneous foetal to adult transition in migratory phenotype that foetal fibroblasts undergo after approximately 50–55 population doublings in vitro is correlated with a cessation of MSF production; (2) breast cancer patient fibroblasts do not undergo such a phenotypic transition and continue to produce MSF for their entire in vitro lifespan. These foetal-like cancer patient fibroblasts do, however, resemble normal adult cells by a number of other criteria, including population doubling potential, enhanced migration in the presence of serum compared to platelet-poor plasma, saturation cell density and morphology in confluent culture. These data indicate that the fibroblasts of breast cancer patients express a mixture of both foetal and adult phenotypic characteristics. Such a finding is consistent with published information indicating that foetal-to-adult transitions in various fibroblast phenotypic characteristics occur in a temporally disparate fashion during normal development, and further imply that cancer patient fibroblasts have undergone only certain of these transitions.
The structure and interactions of components of nuclear envelopes from Xenopus oocyte germinal vesicles observed by heavy metal shadowing
Authors: Stewart Murray , Whytock Sue
Published: April 2021

We have examined the structure of the nuclear envelope of oocytes of Xenopus laevis by electron microscopy of metal-shadowed specimens. Material was prepared by either freeze-drying or a rapid protocol using air-drying after dehydration in ethanol followed by amyl acetate. These methods emphasized different aspects of the structure and enabled an integrated view of the arrangement of nuclear pore complexes, nuclear lamina and pore-connecting fibrils to be assembled. In specimens prepared by either airdrying or freeze-drying, the lamina meshwork beneath the nuclear face of the envelope was well preserved, but the fine structure of the nuclear pores was superior in freeze-dried preparations. Both methods also showed pore-connecting fibrils that were clearly not components of the lamina. By using stereo pairs, we established criteria for recognizing the cytoplasmic and nucleoplasmic faces of shadowed nuclear envelopes. These views also enabled us to identify the levels at which different fibrous components were attached to the pores. In particular, we were able to visualize the nuclear lamina fibres and poreconnecting fibrils simultaneously and show that they attach to the pore complexes at different levels. We supplemented this work by using a range of treatments to disrupt the nuclear envelope slightly and gained several insights into this structure as a result. Sometimes pore complexes and their connecting fibrils were stripped from the envelope. This enabled a clearer view of these connections to be obtained without the lamina present. Moreover, in some conditions, the nuclear pore complexes and fibrous lamina began to disintegrate, thereby showing some of the morphological components from which they were assembled.
Deficiency in immune complex uptake by chronic granulomatous disease neutrophils
Authors: Petty Howard R. , Franc Joseph W. , Boxer Laurence A.
Published: April 2021

Receptor-mediated binding and endocytosis by normal and chronic granulomatous disease (CGD) polymorphonuclear (PMN) leucocytes have been investigated. Our results suggest that activation of the respiratory burst is a requisite for normal immune complex internalization. Cell-associated immune complexes were detected by fluorescence microscopy, electron microscopy and flow cytometric techniques. These measurements indicate that immune complex uptake is defective in CGD patients, but not in normal subjects. Antibody-dependent binding and complement-dependent binding and endocytosis were not affected. The functional association of the respiratory burst and antibody-dependent endo cytosis was further explored using rhodamine-labelled glucose oxidase: anti-glucose oxidase immune complexes. These complexes generate H2O2 in the presence of glucose. When treated with these complexes, CGD PMNs showed similar glucose-dose-dependent increases in both H2O2 production and cell-associated fluorescence. Therefore: (1) respiratory burst and immune complex uptake are coexisting deficiencies that may be a manifestation of a single defect; and (2) the respiratory burst may be one participant in immune complex endocytic triggering.
The role of intracellular Ca2+ during early sexual development in Dictyostelium discoideum: effects of laci3, lns(1,4,5)P3, TMB-8, chlortetracycline and A23187 on cell fusion
Authors: Lydan Michael A. , O’Day Danton H.
Published: April 2021

The agents LaCl3, Ins(l,4,5)P3, TMB-8, chlortetracycline (CTC) and A23187 were used to study the requirement for internal calcium mobilization during gamete cell fusion in Dictyostelium discoideum. The inhibition of the influx of calcium (LaCl3) prevented cell fusion in a dose-dependent manner. At the intracellular level, Ins(l,4,5)P3, an endogenous regulator of calcium release from intracellular stores, stimulated cell fusion within one hour following its addition. Treatment with agents that prevent the release of calcium from intracellular stores (TMB-8, CTC) also inhibited cell fusion in a dose-dependent manner. However, the non-specific augmentation of cytosolic calcium levels through the use of the ionophore A23187 inhibited cell fusion, and the amount of inhibition was directly related to the drug concentration. Studies on cell morphology and growth plus results from reversibility experiments involving the ability to form macrocysts reveal that these effects are not due to non-specific drug toxicity. In total, these results suggest that the mobilization of calcium both from the extracellular environment and from intracellular stores is important and is probably regulated during gamete cell fusion in D. discoideum.
Insulin-like growth factors and the multiplication of Tera-2, A human teratoma-derived cell line
Authors: Biddle C. , Li C. H. , Schofield P. N. , Tate V. E. , Hopkins B. , Engstrom W. , Husklsson N. S. , Graham C. F.
Published: April 2021

A human teratoma cell line (Tera-2) was grown in serum-free medium, and the population multiplication was stimulated by the addition of somato-medins/insulin-like growth factors (IGFs). Both IGF-I and IGF-II gave maximal stimulation when added daily at 10 ng ml-1. The IGFs did not substantially change the labelling index of the cells, and the IGFs appeared to exert their effect on population multiplication by increasing cell survival. Membranes isolated from Tera-2 cells displayed both type 1 and type 2 IGF receptors.
Endoplasmin is a reticuloplasmin
Authors: Koch G. L. E. , Macer D. R. J. , Wooding F. B. P.
Published: April 2021

The location of endoplasmin in the endoplasmic reticulum was investigated by biochemical and immunoelectron microscopic analyses. The protein could be obtained in a soluble form by procedures that do not involve the use of any detergents. The soluble protein has the amino- and carboxy-terminal sequences of the intact molecule, showing that it has not been proteolysed. Application of the Triton X-114 phaseseparation test does not reveal significant hydrophobicity in the molecule. Immunogold labelling studies on cells with a dilated endoplasmic reticulum (ER) lumen show that endoplasmin is uniformly distributed throughout the lumen, with no evidence of a preferential association with the membrane. These studies clearly demonstrate that endoplasmin is a luminal protein of the ER, i.e. a reticuloplasmin, and not an integral membrane protein.
The ultrastructure and timing of events in the nuclear cycle of the fungus Entomophaga aulicae
Authors: Murrin Faye , Newcomb William , Heath I. Brent
Published: April 2021

The ultrastructure of the mitotic nuclear division cycle of the fungus Entomophaga aulicae was studied from serial sections of hyphal tips and protoplasts. The extranuclear bar-shaped nucleus-associated organelle (NAO) remained associated with the persistent nuclear envelope throughout. Prior to spindle formation, a patch of intranuclear NAO-associated chromatin detached from the nuclear envelope to yield a chromatinfree zone containing fine filaments and a linear array of presumptive kinetochores. Early metaphase spindles less than 1 pm in length were characterized by a ‘fused’ metaphase plate consisting of kinetochore-associated chromatin and a full complement of at least 15 kinetochore microtubules per half-spindle, while most of the chromatin was remote from the intranuclear spindle. Analysis of the distribution of antiparallel spindle microtubules indicated that polar separation and concomitant spindle elongation through metaphase were not accompanied by intermicrotubule sliding. Anaphase exhibited extensive decondensation of the large patches of condensed chromatin characteristic of all other stages. In a logarithmically growing protoplast population all nuclei contained spindle microtubules, with metaphase occupying approximately 66% of the nuclear cycle time. The calculated genome size of 4-3 pg, and average DNA content per chromosome of 0-3 pg, are extremely high for fungi.
Differential Cytolocalization Of Prosomes In Axolotl During Oogenesis And Meiotic Maturation
Authors: Gautier J. , Pal J. K. , de Sa M.-F. Grossi , Beetschen J. C. , Scherrer K.
Published: April 2021

The prosomes, a novel type of small RNA-protein complex previously characterized in avian and mammalian cells, were isolated from axolotl (Ambystoma mexicanutn) oocytes and identified by sedimentation analysis and protein composition. The prosomal nature of these particles was further ascertained by immunoblot analysis with anti-duck prosome monoclonal antibodies. By in vitro [3SS] methionine labelling, de novo synthesis of prosomal proteins could be detected neither during oogenesis nor meiotic maturation. The results obtained by both indirect immunofluorescence and immunoblot analyses demonstrated a dramatic change in the localization of prosomal antigens during oocyte development. They were initially detected in the oocyte cytoplasm, during oocyte growth. At the end of vitellogenesis (stages V-VI), they entered the nucleus (germinal vesicle) and were accumulated there to the highest concentration. During oocyte maturation, after nuclear envelope breakdown, prosomal antigens were found to be localized again in the cytoplasm, until fertilization. No specific localization of prosomal antigens in mature oocytes, unfertilized and fertilized eggs was observed within the oocyte cytoplasm in relation to the cytoplasmic rearrangements leading to grey crescent formation.
Tubulin Post-Translational Modifications And The Construction Of Microtubular Organelles In Trypanosoma Brucei
Authors: Sasse Rosemary , Gull Keith
Published: April 2021

We have used specific monoclonal antibodies to facilitate a study of acetylated and tyrosinated α-tubulin in the microtubule (MT) arrays in the Trypanosoma brucei cell. Acetylated α-tubulin is not solely located in the stable microtubular arrays but is present even in the ephemeral microtubules of the mitotic spindle. Moreover, there is a uniform distribution of this isoform in all arrays. Studies of flagella complexes show that acetylation is concomitant with assembly of MTs. There is no subsequent major modulation in the content of acetylated α-tubulin in MTs. Conversely, polymerizing flagellar MTs have a high tyrosinated α-tubulin content, which is subsequently reduced to a basal level at a discrete point in the cell cycle. The MTs of the intranu-clear mitotic spindle appear never to contain tyrosinated α-tubulin, suggesting that they are actually constructed of detyrosinated α-tubulin or that detyrosination is extremely rapid at this time in the cell cycle. T. brucei therefore, represents a cell type with extremely active mechanisms for the post-translational modification of α-tubulin. Our analyses of the timing of acquisition and modulation in relation to MT construction in T. brucei, suggest that acetylation and detyrosination of α-tubulin are two independently regulated post-translational modifications, that are not uniquely associated with particular subsets of MTs of defined lability, position or function. Post-assembly detyrosination of α-tubulin may provide a mechanism whereby the cell could discriminate between new and old MTs, during construction of the cytoskeleton through the cell cycle. However, we also suggest that continuation of detyrosination, allows the cell, at cell division, to partition into daughter cells two equivalent sets of cytoskeletal MTs.
The Role Of Hmg Coa Reductase And Dolichol Synthesis In The Control Of 3t6 Cell Proliferation: Effects Of Cell Crowding, Serum Depletion And Addition Of Epidermal Growth Factor
Authors: Larsson Olle
Published: April 2021

The proliferation of 3T6 cells was substantially decreased when the monolayer cultures were allowed to reach confluency. This growth inhibition (so-called density-dependent inhibition) was of the same magnitude as that following serum depletion in non-confluent cultures. Each type of growth inhibition was correlated to a depression of the activity of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase, an enzyme that regulates the biosynthesis of cholesterol and isoprenoid derivatives (e.g. dolichol) by catalysing the reduction of HMG CoA (which is derived from acetyl-CoA) into mevalonate. However, the depression of enzyme activity was more substantial in cells exposed to cell crowding than that in serum-depleted cells (87 and 48 %, respectively). On the other hand, there was a 60–65 % inhibition of the incorporation of mevalonate into dolichol due to serum deprivation, while it remained at normal level in confluent cultures, which implies that the inhibitory effects on dolichol synthesis due to these two experimental conditions were approximately equipotent. Addition of epidermal growth factor (EGF) to the cell cultures, whose proliferation was inhibited due to serum depletion, restored DNA synthesis completely, and these effects were related to anormalization of the activity of HMG CoA reductase and of the incorporation of mevalonate into dolichol. In contrast, in confluent cells addition of EGF only caused a slight increase in DNA synthesis and activity of HMG CoA reductase, and there was no significant increase in the incorporation of mevalonate into dolichol either. Supplementation with mevalonate as well as dolichol to confluent cell cultures induced a significant increase in DNA synthesis, while only dolichol was capable of increasing DNA synthesis in serum-depleted cells.

Taken together, these results propose: (1) the existence of two regulatory sites in the biosynthesis of dolichol, the first one of which is constituted by HMG CoA reductase, and the second one by an unknown enzyme in the distal part of the pathway; (2) that the activity of HMG CoA reductase is stimulated by serum and growth factors, and is inhibited by cell crowding; (3) that the second step is independent of the culture density, but is stimulated by serum and growth factors; (4) that these two regulatory sites are involved in the control of cell proliferation in 3T6 cells.
Effects Of Phorbol Esters On Shape And Locomotion Of Human Blood Lymphocytes
Authors: Wilkinson P. C. , Lackie J. M. , Haston Wendy S. , Islam Laila N.
Published: April 2021

The effects of phorbol esters on shape change and locomotion of human blood lymphocytes were studied both immediately after separating the cells from blood and after overnight culture. Phorbol myristate acetate (PMA), phorbol dibu-tyrate (PDB) and related esters produced complex shape changes in lymphocytes at both times. These shapes were analysed quantitatively using objective measurements derived from the moments of cell shapes. Immediately after removal from blood, many lymphocytes (20–60% of the total) protruded and retracted veils or spikes at more than one point on the cell surface. The morphology of these cells was not typical of locomotor cells. Usually, formation of a veil was not followed by a contraction wave moving down the cell, though some cells did show contraction waves, and some moved into collagen gels or filters. After overnight culture, a high proportion (70–80 %) of cells had changed shape in PMA and PDB. Although the shapes were still atypical, they resembled classical locomotor morphology moreclosely; veils formed at one point on the cell surface tended to persist, and contraction waves and constriction rings were seen in many cells. These cells moved in large numbers into collagen gels or filters. Comparison of the paths traversed by PMA-treated lymphocytes in collagen gels suggested that cells cultured in PMA for 24 h pursued more persistent paths than those in short-term culture, but the difference was not marked. We suggest that phorbol esters induce immediate shape change without inducing the complete sequence of motor events necessary for efficient locomotion, whereas after prolonged culture in phorbol esters, locomotion is more efficient, possibly because phorbol esters, like other growth activators, stimulate events during the G| phase of growth that are necessary for full expression of locomotor capacity.
Contact-mediated triggering of lamella formation by Dictyostelium amoebae on solid surfaces
Authors: Owens Norman F. , Gingell David , Bailey Juliet
Published: April 2021

Amoebae of the slime mould Dictyostelium discoid-eum form broad ultrathin cytoplasmic lamellae by a centripetal contractile process soon after they have spread on certain solid surfaces. We have investigated the surface requirements for initial triggering of this contact-mediated signalling system. The lamellar response is not normally evoked by glass, but is seen on glass covalently derivatized with paraffinic chains, as well as on glass covalently derivatized with amine groups and on glass bearing adsorbed polylysine. We have recorded the frequency of the lamellar response on these surfaces as a function of ionic strength and pH, and have measured the electrostatic potentials of the surfaces by the streaming potential method. Using these data we have concluded that the general trigger for the lamellar response is not a ‘simplE′ physical or chemical property of the substrata : it is not dependent on specific chemical groups, degree of hydrophobicity, electrostatic potential, or charge density, taken as isolated factors. It seems likely that triggering is dependent on the overall energetics of cell-substratum interaction.
Intermicrotubular actin filaments in the transalar cytoskeletal arrays of Drosophila
Authors: Mogensen M. M. , Tucker J. B.
Published: April 2021

Rabbit muscle myosin subfragment SI decorates 6 nm diameter filaments in Drosophila wing epidermal cells in the arrowhead fashion characteristic of the binding of subfragment SI to actin filaments. The filaments in question are concentrated between microtubules that are mostly composed of 15 protofilaments and form cell surface-associated transcellular bundles. There are indications that the majority of the actin filaments have the same polarity and that, like the microtubules, they may elongate from sites at the apical surfaces of the cells.

The bundles of F actin and microtubules occur in dorsal and ventral epidermal cell layers of a wing blade. They are joined in dorso-ventral pairs by attachment desmosomes. These transalar cytoskeletal arrays may provide an example of a situation where actin filaments operate as stiffeners rather than active generators of force in conjunction with myosin. The arrays probably function as non-contractile pillars to maintain basal cell extensions and keep haemocoelic spaces open in the highly folded and expanding wing blades of late pupae.
Changes in the number and volume of fibrillar centres with the inactivation of nucleoli at erythropoiesis
Authors: Zatsepina Olga V. , Chelidze Pavel V. , Chentsov Yuri S.
Published: April 2021

The number and volume of fibrillar centres, the structural components of interphase cell nucleoli on the surface of which rRNA is synthesized, have been studied in differentiating erythroblasts of mouse embryo liver. Complete series of ultrathin sections of erythroblast nuclei have been used at the main stages of differentiation: proerythroblast, basophilic erythroblast, polychromatophilic erythroblast and normoblast. It has been shown that in the active nucleoli of proerythroblasts the number of fibrillar centres is correlated with cell ploidy and exceeds by several-fold the number of nucleolusorganizing regions of chromosomes. The total volumes of fibrillar centres in 2C (0 ·369 μm3) and 4C (0 ·749 μm3) proerythroblasts are proportional to number of nucleolus- organizing regions. With the maturation of erythroblasts the total number of fibrillar centres declines and in normoblasts it is 3-to 10-fold less than that of the nucleolus- organizing regions. The total volume of fibrillar centres in normoblasts (0-102 μm3) is threefold smaller than that in proerythroblasts (0 ·369 μm3), even though the mean volumes of individual fibrillar centres are significantly higher (0·0042 μm3 in proerythroblasts and 0 ·039 in normoblasts). The optical density of fibrillar centres in normoblasts can be higher compared with that of proerythroblasts. It has been suggested that the inactivation of nucleoli at erythropoiesis is accompanied by the fusion of individual fibrillar centres and, possibly, by the compaction of their material.
Ultraviolet microbeam irradiation of chromosomal spindle fibres shears microtubules and permits study of the new free ends in vivo
Authors: Wilson Paula J. , Forer Arthur
Published: April 2021

Irradiation of birefringent chromosomal spindle fibres in cranefly spermatocytes in metaphase I or anaphase I produces an area of reduced birefringence (ARB) on the fibre. This ARB moves poleward and is lost at the pole. Ultrastructural and immunofluorescence analysis of ARBs obtained by irradiation with monochromatic ultraviolet light of wavelength 260 nm shows that the microtubules in the irradiated area are depolymerized, though the rest of the spindle appears unaffected. The area of microtubule depolymerization moves poleward with the ARB, and once the ARB reaches the pole the irradiated half-spindle appears normal. The motion of the ARB, therefore, appears to be due to the behaviour of the sheared microtubules in the chromosomal spindle fibre. The relative stability of the sheared microtubules shows that chromosomal fibre microtubules are not dynamically unstable, as are microtubules under certain conditions in vitro. However, ARB motion may be due to a moderated version of dynamic instability. Possible models for ARB motion are discussed.
Functionally polarized layers formed by epidermal cells on a permeable transparent collagen film
Authors: Yoshizato Katsutoshi , Nishikawa Akio , Taira Toshio
Published: April 2021

Rat epidermal cells were cultured on a transparent collagen film, which was permeable to low Mr substances. Then the cells were bathed in both media (apical and basal), which were separated by the collagen membrane. The cells formed a multilayered epidermal sheet with well-developed structures of desmosomes. This sheet on a permeable support was found to be an effective permeability barrier for glucose and amino acids. The epidermal layer showed functional polarity for the uptake and excretion of nutrients, metabolites and newly synthesized proteins: glucose and amino acids were taken up exclusively from the basal medium and lactate was secreted selectively into the same medium, whereas ammonia was secreted into the apical medium. The apical media became more acidic than the basal ones, presumably due to the preferential distribution of H+ transport systems on the apical side of the epidermal layer. The epidermal cells that expressed functional polarities in vitro as described above were able to proliferate and differentiate, and remained healthy for as long as at least 40 days even using a conventional culture medium with foetal calf serum, but without any special growth factors and feeder cells.
Dissociation and re-assembly of the endoplasmic reticulum in live cells
Authors: Koch G. L. E. , Booth C. , Wooding F. B. P.
Published: April 2021

The endoplasmic reticulum (ER) of a typical interphase 3T3 fibroblast consists of a compact perinuclear arrangement of cistemae and lamellae which can be observed by immunofluorescence with antiendoplasmin. During mitosis the reticulum dissociates into small fragments from which it appears to re-assemble in the daughter cells. When interphase 3T3 cells are exposed to calcium ionophores, but not other ionophores, there is a similar dissociation of the ER into small uniform fragments, which are dispersed throughout the cytoplasm. Electron microscopy shows that the fragments consist of small vesicular structures and that essentially all of the rough ER except the nuclear envelope is dissociated. The dissociation of the ER by calcium ionophore is a relatively specific process since other organelles and supramolecular assemblies remain unaffected. When cells with dis sociated ER are returned to normal medium, there is a rapid reassembly of the fragments into the continuous reticulum. In a proportion of the cells it is possible to observe linear arrays of the fragments, which probably represent intermediates in the re-assembly process. These observations demonstrate that the ER in interphase 3T3 cells can be dissociated into, and re-assembled from, small fragments. Re-assembly of the ER from the fragments is dependent on the presence of millimolar levels of calcium in the external medium. In the presence of calcium, re-assembly is inhibited by the calcium channel blocker, verapamil. Thus calcium ions appear to play an important role in ER structure and assembly.
Microtubule and microfilament distribution during the secretory activity of an insect gland
Authors: Dallai R. , Marchini D. , Callaini G.
Published: April 2021

The distribution of microfilaments and microtubules was studied in the secretory cells of the accessory glands of the medfly (Ceratitis capitata) female reproductive system. Actin, demonstrated by rhodamine-phalloidin and heavy meromyosin decoration, is present around the extracellular storage cavity at the level of microvilli and the subapical cytoplasm of the cell. The size of such a cavity varies in relation to the secretory cycle of the cells and also the distribution of microfilaments. In the early stage of activity microtubules detected using an antibody against a-tubulin are distributed at the periphery of the cell and around the extracellular cavity. During secretion they occupy the whole of the cytoplasm and disappear at the end of the secretory cycle. The mechanism of the discharge of the secretion from the extracellular cavity is discussed.
Localized osmotic swelling and cell fusion in erythrocytes: Possible implications for exocytosis
Authors: Ahkong Quet F. , Lucy Jack A.
Published: April 2021

Factors that govern the experimentally induced fusion of erythrocytes with one another may generally be relevant to whether or not osmotic forces drive membrane fusion in exocytosis because, under appropriate conditions, osmotic swelling can drive the fusion of erythrocytes. It is now reported that these cells fuse when they are subjected to osmotic swelling caused by exposure to small permeant molecules. The behaviour of erythrocytes in fusion induced by treatment with a concentrated solution of high molecular weight poly(ethylene glycol) (PEG) is also of specific interest in relation to exocytosis because the haemoglobin of erythrocytes that are dehydrated by concentrated solutions of the polymer may be regarded as a model for the tightly packed, dehydrated contents of the granules in secretory cells. We have observed that, under certain conditions of rehydration, the swelling of aqueous microdroplets between the dehydrated haemoglobin and the plasma membrane is closely associated with the fusion of partially rehydrated but still shrunken, PEG-treated erythrocytes. It is therefore apparent that osmotic forces, acting locally at the sites of aqueous microdroplets, can drive the fusion of membranes that encapsulate a dehydrated, concentrated protein, even though gross osmotic swelling at the level of the light microscope is absent. This finding is consistent with the possibility that osmotic swelling may play a role in exocytotic membrane fusion if it is restricted to a small zone immediately under the granule membrane.
Gradient and temporal signal perception in chemotaxis
Authors: Vicker Michael G.
Published: April 2021

In a recent Commentary, Lauffenburger et al. (1988) argued that chemotaxis depends on the perception of a chemoattractant (CA) concentration (CCA) gradient across the length of a polymorphonuclear (PMN) neutrophil leucocyte. Haston & Wilkinson (1987) agreed with this view, pointing out that PMN polarize after contact with a CA. In the light of the important role expected of concentration gradients in taxis and in morphogenesis, it seems appropriate to continue this dialogue; first, by replying to the remarks of Lauffenburger et al. (1988) about our failure to detect taxis in spatial gradients and, second, by introducing into it some rudimentary facts about cell behaviour and signals, which have apparently attracted little attention in discussions of gradient perception.
Ki-67 detects a nuclear matrix-associated proliferation-related antigen : I. Intracellular localization during interphase
Authors: Verheijen R. , Kuijpers H. J. H. , Schlingemann R. O. , Boehmer A. L. M. , Driel R. Van , Brakenhoff G. J. , Ramaekers F. C. S.
Published: April 2021

Ki-67 is a commercially available mouse monoclonal antibody, which reacts with a nuclear antigen in proliferating cells. The antibody can be used to determine the growth fraction of human tumours in situ and has been shown to be of prognostic importance.

In this study it is shown that in interphase cells Ki-67 reacts with an antigen, mainly present in the nucleoli. Confocal scanning laser microscopy and immunoelectron microscopy on human MR65 monolayer cells revealed that this nucleolar antigen is predominantly localized in the nucleolar cortex and in the dense fibrillar components.

The Ki-67 antigen appeared to be preserved in nuclear matrix preparations obtained after in situ fractionation of MR65 cells. Despite many efforts, we could not identify the antigen in immunoblotting or immunoprecipitation assays.

Testing of cell cultures of different species by means of indirect immunofluorescence revealed that the antibody reacted with human cells and with the Rhesus monkey kidney-derived cell line LLC-MK2.