bluuebunny/crossref_metadata_2025 huggingface dataset:
Polymorphism of tubulin assembly in vitro
Authors: Karecla Paula , Hirst Elizabeth , Bayley Peter
Published: April 2021

Polymorphism in the self-assembly of tubulin dimer and microtubule protein (tubulin plus the microtubule-associated proteins) has been investigated as a function of systematic variation of solution composition (i.e. buffer ion, [glycerol] and [Mg2+]). The nature of the assembly product was examined using negative staining and thin sectioning electron microscopy.

The morphology of the product of assembly of tubulin dimer was found to be strongly influenced by the concentration of glycerol and Mg2+ in Pipes and Mes buffers; the effects are less marked in phosphate buffer. Formation of bona fide micro-tubules in 0.1 M-Pipes occurs for a limited range of solution conditions (e.g. with [glycerol] <2 M and [Mg2+]<l mM). Conditions of elevated [glycerol] and [Mg2+], which enhance the rate and extent of assembly, have the adverse effect of strongly promoting the formation of polymorphic forms in addition to, and in place of, the normal microtubule morphology.

In both Pipes and Mes buffers, increasing [glycerol] from 1 to 3 M favours the formation of extended multiply curved sheets, apparently made up from a basic structure with an S-like cross-section. By contrast, increasing [Mg2+] promotes the formation of junctions between microtubule walls, giving products whose cross-section shows multiple hook-like appendages, attached to closed microtubules. The assembly of tubulin dimer in a typical ‘dimer assembly buffer’ (e.g. 0.05-0.1 M-Mes, with 1-3.4M-glycerol and 2–7mM-Mg2+), invariably produces substantial proportions of non-microtubule structures such as open sheets, ribbons, and hooked structures.

We conclude that the self-assembly of tubulin dimer exclusively into bona fide microtubules occurs over a very restricted range of solution conditions in the normally used Pipes- and Mes-based buffers. Deviation from these conditions readily promotes the formation of mixtures of polymorphic forms. Many buffer systems used for the assembly and disassembly of microtubules composed of tubulin dimer appear likely to promote the formation of structures related to, but significantly different from, normal microtubules. This represents a cautionary factor in the interpretation of in vitro assembly and disassembly properties of microtubules.
A novel component of the axonal cortical cytoskeleton, A60, defined by a monoclonal antibody
Authors: Rayner Denise A. , Baines Anthony J.
Published: April 2021

A Mr 60 000 protein of the axonal cortical cytoplasm, which is recognized by a novel monoclonal antibody, is described. The antibody, DR1, was produced by immunizing mice with a soluble extract of bovine brain membranes that is enriched in known membrane cytoskeletal proteins. DR1 recognizes a Mr60000 protein in this extract. Immunofluorescence and subcellular fractionation reveal that the protein is primarily located in axons, where it appears to form a thick lining to the axolemma. Operationally, this Mr 60000 protein is defined as a cytoskeleton-associated peripheral membrane protein. It is solubilized from brain membranes only under harsh conditions (0.1M-NaOH), but not with KI (0.8M) or Triton X-100 (1%). It is present at higher levels in the central nervous system than in peripheral nerves that have been examined. The Mr 60 000 protein copurifies with neurofilaments through cycles of assembly and disassembly. It does not appear to react with the anti-IFA antibody, suggesting that it is not a member of the intermediate filament class of proteins. This Mr60000 protein, which we designate A60, is distinct from other known neurofilament-associated proteins, including the Mr 60 000 protein α-internexin and the Mr 58000 intermediate filament protein peripherin. A60 is suggested as being a previously unrecognized component of the axonal cortical cytoskeleton.
Regulatory signals affecting a selective loss of mRNA in Dictyostelium discoideum
Authors: Hassanain Hamdy H. , Kopachik Will
Published: April 2021

We identified signals that affect mRNA levels complementary to a gene that is highly expressed in vegetative Dictyostelium discoideum cells. This gene has been cloned as cDNA in the plasmid pcD-D2. The level of transcripts homologous to pcD-D2 fell dramatically in strain XP55 during the aggregation stage of development when cells differentiate on agar. The level, however, did not fall simply as a result of starvation or aggregationspecific cell contact. Rather, before the level is reduced cells must be deprived of amino acids and cyclic AMP administered in amounts and at intervals in pulses to mimic cyclic AMP signal-relay in aggregation. This effect can be blocked either with cyclic AMP-S (a non-hydrolysable cyclic AMP analogue) or adenosine, both of which prevent cyclic AMP binding to the cyclic AMP cell surface receptor. It is also blocked in ‘frigid’ aggregationdeficient mutants HC85 and HC112 known to be defective in a Gα protein. We conclude that the transcript level is balanced by positive nutritional signals acting against negative signals transduced in part through a cell surface cyclic AMP receptor.
Differences between adult and foetal fibroblasts in the regulation of hyaluronate synthesis: correlation with migratory activity
Authors: John Chen W. Y. , Grant Michael E. , Schor Ana M. , Schor Seth L.
Published: April 2021

We have previously reported that confluent foetal fibroblasts migrate into three-dimensional collagen gel matrices to a significantly greater extent than do adult cells. Hyaluronic acid (HA) is a major constituent of the extracellular matrix deposited by fibroblasts and has been demonstrated to stimulate the migration of a number of different cell types. Previous studies have indicated that the synthesis of HA by normal adult skin fibroblasts declines significantly when the cells achieve confluence. Data presented in this paper indicate that foetal fibroblasts differ from adult cells in this respect, in that they do not show an inverse relationship between cell density and HA synthesis, i.e. confluent foetal fibroblasts continue to produce approximately the same amount of HA as do subconfluent cells. These data suggest that the synthesis of relatively high levels of HA by foetal fibroblasts at confluence may be causally related to the elevated migration displayed by these cells. In this context, a close correlation was observed between the level of HA synthesized by confluent foetal and adult fibroblasts and the differential migratory activity displayed by these cells. Such differences in HA synthesis and migratory behaviour were only apparent at cell confluence, with subconfluent foetal and adult fi-broblasts being indistinguishable in terms of these two criteria. Our data further reveal that: (1) cell density affects the size class of HA synthesized by both foetal and adult cells; and that (2) there is a considerable degree of heterogeneity amongst the nine different fibroblast lines examined in this study in terms of the size class of HA that they produce.
Action of activated 27 000 M
r toxin from Bacillus thuringiensis var. israelensis on Malpighian tubules of the insect, Rhodnius prolixus
Authors: Maddrell S. H. P. , Overton J. A. , Ellar D. J. , Knowles B. H.
Published: April 2021

The action of activated 27 000 Mr toxin from Bacillus thuringiensis var. israelensis (Bti toxin) on Malpighian tubules of Rhodnius prolixus has been investigated. Its binding to the tubules is slowed by low temperature but is not prevented even at 0°C. The binding is less effective at pH 10 than at pH 7. Pretreatment of the tubules with 0.1 mmol 1−1 ouabain or bumetanide or 1 μmoll−1 5-hydroxytrypt-amine did not affect the toxicity of the toxin. The toxin causes very large changes in the trans-epithelial potential difference; it changes from 40 mV, lumen negative, often to more than 100 mV, lumen positive. This reflects an initial collapse of the potential of the basal cell membrane, followed by a large positive-going potential change at the luminal cell membrane. Just prior to the effects of the toxin on rapid fluid secretion, the basal cell membrane becomes permeable to sucrose molecules. Raffinose at 170 mmol 1−1 in the bathing solution does not protect the tubules from Bti toxin action but dextran, Mr5000, at 60 mmol 1−1 significantly delayed failure of fluid secretion and, even more, the onset of staining of the tubule cells with Trypan Blue. Exposing tubules to saline that is calcium-free and/or magnesium-free, or has a composition adjusted to be similar to that of the intracellular milieu, does not affect the time course of failure of fluid secretion induced by the toxin. There is no evidence that effective aggregates of Bti toxin molecules are formed in concentrated solutions.

The time courses of failure of fluid secretion induced by Bti toxin and melittin differ. Melittin action also involves a delay before effects are observable, but its effects then develop more slowly than with Bti toxin. This is consistent with the suggestion that melittin and Bti toxin act by forming similarly sized pores but that tetramers of melittin form pores while a larger number of molecules of Bti toxin are required to form a pore.
Unique geometry of actin-membrane anchorage sites in avian gizzard smooth muscle cells
Authors: Draeger A. , Stelzer E. H. K. , Herzog M. , Small J. V.
Published: April 2021

Adherens junctions in isolated avian gizzard smooth muscle cells appear as short longitudinal streaks or chevrons that are arranged in periodic, mainly transverse bands along the cell surface. This barrel-like geometry, revealed by antibodies to either vinculin or talin, was seen also in teased gizzard strips by confocal laser-scanning microscopy and contrasted with the rib-like surface pattern observed here and previously in other avian and mammalian smooth muscles. There were on average 67 transverse bands per gizzard cell and an estimated total of around 800 vinculin/talin sites. The longitudinal spacing between the transverse bands of vinculin streaks in the gizzard cells changed from 4–5 μm in extended cells to around Iμ m in shortened cells and the bands remained essentially transverse at all cell lengths, inconsistent with a screw-like mode of cell shortening as has been invoked for smooth muscle cells by others. The absence of rotation on shortening was confirmed by observations on isolated and bead-decorated skinned cells that were induced to contract with ATP. Counterlabelling of cells with α-actinin antibodies produced more or less exclusive staining of the cytoplasmic dense bodies, and little surface label: the total number of dense bodies per cell, estimated from confocal microscope through focal series was in the range of 3000. The data are consistent with a periodic anchorage of actin filaments to the cell surface and, in turn, with the existence of regularly spaced contractile assemblies.
Further characterization of the antigen defined by the monoclonal antibody m27
Authors: Bramwell Michael E. , Humm Susan M.
Published: April 2021

Using immunoblotting techniques, the antigen that binds the monoclonal antibody M27 has been clearly defined in terms of apparent molecular mass and distribution. In reducing conditions it has an apparent mass of 178K (K= 103 Mr) and is present in the cytoplasm and membranes of all mammalian tissue culture cells so far examined. It is absent from lines derived from avian, piscine and amphibian sources. It is also absent from foetal liver of both rat and mouse, but subsequently appears after cultivation in vitro. Similarly, it can be detected on rat lymphocytes only after mitogenic stimulation. However, it is found on both hepatoma and lymphoma cells in vitro, and on in vivo tumours from murine sources. It thus appears to be associated with cell proliferation.
Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei
Authors: Woodward R. , Gull K.
Published: April 2021

We have used irnmunofluorescent detection of 5-bromo-2-deoxyuridine-substituted DNA in order to determine the timing of initiation and the duration of nuclear and kinetoplast S-phases within the procyclic stage of the Trypanosoma brucei cell cycle. Both nuclear and kinetoplast S-phases were shown to be periodic, occupying 0.18 and 0.12 of the unit cell cycle, respectively. In addition, initiation of both of these S-phases were in approximate synchrony, differing by only 0.03 of the unit cell cycle. We have also used a monoclonal antibody that recognises the basal bodies of T. brucei in order to visualise cells possessing a new pro-basal body and hence determine the time of pro-basal body formation within the cell cycle. Pro-basal body formation occurred within a few minutes of the initiation of nuclear S-phase, at 0.41 of the unit cell cycle. This provides detection of the earliest known cell cycle event in T. brucei at the level of the light microscope. Cell cycle events including initiation of nuclear and kinetoplast DNA replication and pro-basal body formation may be strictly coordinated in T. brucei in order to maintain the precise single-mitochondrion (kinetoplast), singleflagellum status of the interphase cell.
Microtubules and their protofilaments in the flagellum of an insect spermatozoon
Authors: Afzelius B. A. , Bellon P. L. , Lanzavecchia S.
Published: April 2021

Spermatozoa of stick insects have nine accessory tubules, which surround the nine outer microtubular doublets and the two inner microtubular singlets. When fixed in a fixative that was designed to minimize protein denaturation (glutaraldehyde and tannic acid, no osmium post-fixation but block staining with uranyl acetate in water) the accessory tubules were seen to contain 17 protofilaments of the same type as those in the 9+2 microtubular doublets and singlets. The protofilaments in accessory tubules and other microtubules were roughly triangular. When studied by Markham’s photographic method a somewhat different tilt of the two longer sides was seen; this makes it possible to distinguish a polarity in the microtubules, i.e. to differentiate between a microtubule that is viewed from its (—)end to its (+)end from one that is viewed in the opposite direction. The dynein arms of the doublets can be used as an independent type of marker for the polarity. In a computer-aided analysis of the fine structure of the tail axoneme, the A-subtubules of the outer doublets were seen to be not quite equidistant; rather, there were somewhat widened electron-dense interspaces in the ring of protofilaments in four places. The locations of these widened interspaces coincide with the attachment sites for the spoke, the inner dynein arm, the outer dynein arm, and the intertubular material. It is tentatively concluded that proteins of these structures, and perhaps also other microtubule-assocIated proteins, may be anchored deep within the wall of a microtubule rather than just superficially along it.
The predicted amino acid sequence of a centrosphere protein in dividing sea urchin eggs is similar to elongation factor (EF-1a)
Authors: Kuriyama Ryoko , Savereide Paul , Lefebvre Paul , Dasgupta Santanu
Published: April 2021

Monoclonal antibody (SU5), prepared from isolated mitotic spindles of sea urchin eggs, stained centrospheres preferentially and recognized a 5OK (K=103Mr) polypeptide on immunoblots. Three positive clones were isolated by screening a λtll cDNA expression library prepared from sea urchin egg mRNA with SU5. One clone containing a 1.8-kb (1 kb= 103 base-pairs) insert was selected for further characterization. The β-galactosidase fusion protein encoded by the cDNA clone had an apparent relative molecular mass of 150K, indicating that the inserted cDNA produced an estimated 34K of polypeptide. A single 2.2-kb RNA transcript was detected in sea urchin embryos using the cDNA clone as a probe. The cDNA fragment was sequenced and the nucleotide sequence was used to predict the amino acid sequence of the open reading frames in the clone. The putative gene product shows striking similarity to the peptide chain elongation factor (EF-lα) from yeast, fungus, shrimp, insect, mouse and human.
Binding of hexabrachion (tenascin) to the extracellular matrix and substratum and its effect on cell adhesion
Authors: Lightner Virginia A. , Erickson Harold P.
Published: April 2021

Hexabrachion is a large glycoprotein of the extracellular matrix (ECM) that is prominent in embryogenesis, wound healing and tumorigenesis. Because of the role of extracellular matrix proteins in the regulation of cell differentiation and migration, the interaction of hexabrachion with cells as well as with other components of the ECM is of great interest. Early reports suggested that hexabrachion does not bind to fibronectin or gelatin but does bind to chondroitin sulfate proteoglycans. However, more recent reports have suggested that hexabrachion binds to fibronectin and inhibits cell adhesion as well as cell migration on fibronectin.

We have found no evidence of strong hexabrachion-fibronectin binding on either a solid-phase ELISA assay or in a fluid-phase sedimentation assay in which the reactants were allowed to dissociate. However, hexabrachion sedimentation was accelerated in a gradient containing fibronectin throughout. This demonstrates an association between hexabrachions and fibronectin, but the complex is apparently weak and readily reversible. The solid phase ELISA also shows no evidence of hexabrachion binding to gelatin, laminin or types I, III, TV or V collagen.

Hexabrachion does not support strong cell attachment of the cell lines tested. Moreover, hexabrachion can inhibit cell attachment to fibronectin. We demonstrate here that this inhibition requires the hexabrachion to be able to bind to the plastic substratum. The results suggest that hexabrachion inhibition is via a steric inhibition. When the hexabrachion molecules bind to the plastic, they cover up a significant fraction of the underlying fibronectin molecules. Antibody studies are presented that show that hexabrachion can nonspecifically block access of immunoglobulin G molecules to the underlying matrix. This steric blocking is not unique to hexabrachion.
Nerve growth in a small applied electric field and the effects of pharmacological agents on rate and orientation
Authors: McCaig Colin D.
Published: April 2021

The rate of growth and orientation of embryonic Xenopus nerves exposed to pharmacological agents, to an applied electric field or to both simultaneously were studied. The adenyl cyclase activator forskolin (100μM) induced a threefold increase in the rate of elongation, as did an electric field alone. Together, their effect in augmenting rate of growth was additive, but only at a concentration of 50μM forskolin. The normal pattern of faster growth towards cathode than anode was not present in nerves treated with the lectin concanava lin A, which also inhibits normal turning behaviour towards the cathode. Nerve orientation towards the cathode and augmented rates of growth were found in the presence of forskolin or ganglioside GM1. It is suggested that a combined approach of drug treatment and an applied electric field may be useful in promoting nerve regeneration.
Collagen fibrillogenesis in vitro: interaction of types I and V collagen regulates fibril diameter
Authors: Birk David E. , Fitch John M. , Babiarz Joanne P. , Doane Kathleen J. , Linsenmayer Thomas F.
Published: April 2021

The small-diameter fibrils of the chick corneal stroma are heterotypic, composed of both collagen types I and V. This tissue has a high concentration of type V collagen relative to other type I-containing tissues with larger-diameter fibrils, suggesting that heterotypic interactions may have a regulatory role in the control of fibril diameter. The interactions of collagen types I and V were studied using an in vitro self-assembly system. Collagens were purified from lathyritic chick embryos in the presence of protease inhibitors. The type V collagen preparations contained higher molecular weight forms of the α1 (V) and α2(V) chains constituting 60-70% of the total. Rotary-shadow electron micrographs showed a persistence of a small, pepsin-sensitive terminal region in an amount consistent with that seen by electrophoresis. In vitro, this purified type V collagen formed thin fibrils with no apparent periodicity, while type I collagen fibrils had a broad distribution of large diameters. However, when type I collagen was mixed with increasing amounts of type V collagen a progressive and significant decrease in both the mean fibril diameter and the variance was observed for D periodic fibrils. The amino-terminal domain of the type V collagen molecule was required for this regulatory effect and in its absence little diameter reducing activity was observed. Electron microscopy using collagen type specific monoclonal antibodies demonstrated that the fibrils formed were heterotypic, containing both collagen types I and V. These data indicate that the interaction of type V with type I collagen is one mechanism modulating fibril diameter and is at least partially responsible for the regulation of collagen fibril formation.
Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA) structural conservation and the detection of a nucleolar form
Authors: Waseem Naushin H. , Lane David P.
Published: April 2021

The proliferating cell nuclear antigen, PCNA, has recently been identified as the polymerase ô accessory protein. PCNA is essential for cellular DNA synthesis and is also required for the in vitro replication of simian virus 40 (SV40) DNA where it acts to coordinate leading and lagging strand synthesis at the replication fork. The cDNA for rat PCNA was cloned into a series of bacterial expression vectors and the resulting protein used to immunize mice. Eleven new monoclonal antibodies to PCNA have been isolated and characterized. Some of the antibodies recognize epitopes conserved from man to fission yeast. Immunocytochemical analysis of primate epithelial cell lines showed that the antibodies recognized antigenically distinct forms of PCNA and that these forms were localized to different compartments of the nucleus. One antibody reacted exclusively with PCNA in the nucleolus. These results suggest that the PCNA protein may fulfil several separate roles in the cell nucleus associated with changes in its antigenic structure.
Differentiation of human trophoblast cells in vitro as revealed by immunocytochemical staining of desmoplakin and nuclei
Authors: Douglas Gordon C. , King Barry F.
Published: April 2021

The differentiation of isolated human cytotrophoblast cells has been studied by staining cells with anti-desmoplakin antibodies, to reveal cell boundaries, and with anti-nuclear antibodies, to reveal nuclei. During the first 24 h after plating in Ham’s/ Waymouth medium, mononucleated cytotrophoblast cells began to spread and aggregate, forming colonies. This was accompanied by progressive changes in the pattern of desmoplakin staining. In single cells, desmoplakin was dispersed throughout the cytoplasm. As cells aggregated, desmoplakin was redistributed and formed linear, punctate arrays at regions of cell-cell contact, consistent with desmosome formation. A pavement-like staining pattern was maintained even at 5 days. Double staining for desmoplakin and nuclei revealed that most cells within colonies were mononucleated. When plated in a growth medium originally formulated for keratinocytes, cytotrophoblast cells aggregated and formed desmosomes normally. However, after 48 h, cell diameters were increased and nuclei changed from being evenly distributed to forming clusters within large cells, consistent with syncytiotrophoblast formation. While cells grown in Ham’s/Waymouth medium for 2 days could be induced to differentiate by switching to keratinocyte growth medium, cells cultured for 5 days before switching were resistant to the differentiation-inducing effects of the keratinocyte medium. Desmosome-type junctions within colonies of trophoblast cells were unstable and, even after 5 days in culture, could be disrupted by lowering the extracellular Ca2+ concentration. While syncytiotrophoblast formation in keratinocyte growth medium (which contains epidermal growth factor, insulin and hydrocortisone) was accompanied by a 15-to 20-fold increase in chorionic gonadotropin secretion, syncytiotrophoblast formation occurred to a similar extent in keratinocyte basal medium (which does not contain these factors) but with only a twofold increase in chorionic gonadotropin release. These results support the notion that biochemical and morphological differentiation of trophoblast are independent events.
F-actin organization during the cellularization of the Drosophila embryo as revealed with a confocal laser scanning microscope
Authors: Warn R. M. , Robert-Nicoud M.
Published: April 2021

The changes in F-actin organization during the cellularization of the Drosophila embryo have been studied with a confocal laser scanning microscope using fluorescein-phalloidin as a specific stain. Particular study has been made of the changes in the organization of the F-actin network associated with the leading edges of the growing membranes. The role of this actin network in the cellularization process is considered. Other actin-containing structures have also been examined, including the cortical actin layer and a conspicuous region of F-actin aggregates, present beneath the level of the forming cell membranes.
A filamentous network surrounding secretory granules from mast cells
Authors: Nielsen Ellen Holm
Published: April 2021

A freezing-thawing method for rupturing mast cells has made it possible to show that each of the secretory granules is surrounded by a regular network of filaments forming a basket-like structure. The filaments attach to the membrane with hook-like structures. Immunoelectron microscopy of intact mast cells shows that actin occurs mainly in the subplasmalemmal space, whereas in ruptured cells it is seen around granules, indicating that the granule network might consist of actin.
Commentary Signal transduction by the receptors for platelet-derived growth factor
Authors: Heldin Carl-Henrik , Westermark Bengt
Published: April 2021

Platelet-derived growth factor CPDGF) is a connective tissue cell mitogen that originally was purified from human platelets, but recently has been found to be produced by many different cell types (reviewed by Ross et al. 1986; Heldin and Westermark, 1989). The in vivo function of PDGF remains speculative, but the fact that PDGF is released by platelets and by cells involved in the inflammatory reaction and that it stimulates proliferation, chemotaxis and matrix production, suggest a role in tissue repair processes. It is also possible that PDGF is involved in regulation of cell growth and differentiation during embryonal development, since it has been found to be expressed in mouse (Rappolee et al. 1988) and Xenopus (Mercóla et al. 1988) embryos and in human placenta (Goustin et al. 1985). Such a function has been demonstrated in the developing rat optic nerve where PDGF secreted by type-1 astrocytes control the differentiation of O-2A progenitor cells into oligodendrocytes and type-2 astrocytes (Noble et al. 1988; Richardson et al. 1988; Raff et al. 1988). PDGF may also be involved in pathological processes. Thus, unscheduled production of PDGF may account for the excessive cell proliferation seen, e.g., in atherosclerosis and tissue fibrosis, as well as in malignancies. The potent transforming activity of PDGF is illustrated by the fact that the PDGF B chain gene is similar to v-sis, the transforming gene of simian sarcoma virus, and that cell transformation by this virus is exerted by auto-crine action of a PDGF-like factor (reviewed by Wester-mark et al. 1987).
Developmental expression of Drosophila melanogaster small heat-shock proteins
Authors: Haass Christian , Klein Ulrike , Kloetzel Peter-M.
Published: April 2021

We have investigated the developmental expression of the small heat-shock proteins (hsps) during embryogenesis and in adult flies by immunocytology using an antibody that specifically identifies the small hsps. Antibody staining of unstressed early embryos reveals a predominantly cytoplasmic, homogeneous distribution of the small hsps throughout the embryo. At 6h of development small hsp expression can be identified in large, neuroblast-like cells within the extended germ band and in the brain of the embryo. During germ band contraction these cells appear to migrate to the midline where they align pairwise in a segmental pattern. After germ band contraction is complete a high level of small hsp expression can be observed in the midline glia (MECs) and in a cluster of six non-neuronal cells within the midline. In contrast to several other genes that are known to be important for embyogenesis and are expressed in the central nervous system (CNS) of embryos, CNS-specific expression of the small hsps is not restricted to the embryo but is also observed in the adult fly. In adult flies strong small hsp expression is observed in the brain, the thoracic ganglion and the leg nerves. Since the small hsps seem to be expressed predominantly in the glia of the nervous system, our data suggest a protective or stabilizing function of the small hsps within the nervous system during normal fly development, which is independent of the stress response.
Continued DNA synthesis after a mitotic block in the double mutant cut1 cdc11 of the fission yeast Schizosaccharomyces Pombe
Authors: Creanor J. , Mitchison J. M.
Published: April 2021

DNA synthesis is normally dependent on a cell having previously gone through mitosis. Hirano et al. (1986), however, found that DNA synthesis continued at the restrictive temperature in the double mutant cut! cdcll of Schizosaccharomyces pombe even though mitosis was blocked in some of the cells. We have confirmed this result with bulk DNA assays of asynchronous cultures. Synchronous cultures of a diploid double mutant at the restrictive temperature showed two peaks of incorporation with an interval between them that was approximately the same as the doubling time in cell length. Flow cytometry showed that the cells had increased their DNA content from 4C (the diploid value) to about 16C after 7 h. The cytological appearance at this time was mixed, with uninucleate, binucleate and dead cells, but fluorescence measurements on single cells indicated that about half the population had single nuclei with about the 16 C value and had therefore gone through two rounds of DNA synthesis without mitosis.
Cysteine proteinase in Trypanosoma cruzi: Immunocytochemical localization and involvement in parasite-host cell interaction
Authors: Souto-Padrón Thais , Campetella Oscar E. , Cazzulo Juan J. , Souza Wanderley De
Published: April 2021

A monospecific polyclonal antibody obtained against a cysteine proteinase isolated from epimastigotes of Trypanosoma cruzi was used for the immunocytochemical localization of the protein by electron microscopy and to analyse the role played by cysteine proteinase in the process of T. cruzi-host cell interaction. Cytoplasmic structures that correspond to elements of the endosomal-lysosomal (reservosome) system found in epimastigote, amastigote and trypo-mastigote forms reacted intensely with colloidal gold-labelled antibodies using on-section indirect labelling. The surface of most of the tissue culture-derived trypomastigotes was not labelled. However, the flagellar pocket of this form was labelled. All epimastigotes obtained from axenic cultures and amastigote-like forms found in the supernatant of vertebrate cells heavily infected with T. cruzi had their surface intensely labelled, indicating also the surface localization of the protein. Incubation of the parasites in the presence of a sub-agglutinating concentration of the anti-cysteine proteinase antibody led to a marked increase in their uptake by macrophages. In contrast, addition of the F(ab’)2 portion of the same antibody significantly reduced the uptake of the parasites by the macrophages. The results obtained strongly suggest an important participation of cysteine proteinase in the process of T. cruzi- macrophage interaction.
Internalisation of fluorescein isothiocyanate and fluorescein isothiocyanate-dextran by suspension-cultured plant cells
Authors: Cole Louise , Coleman Julian , Evans David , Hawes Chris
Published: April 2021

The uptake of pure non-conjugated fluorescein isothiocyanate (FITC) and of the membrane-impermeant probe FITC-dextran into suspension-cultured carrot cells and protoplasts has been investigated. Commercial samples of a 70K (K=103Mr) FITC-dextran were shown to contain contaminant FITC and/or its degradation products, which were rapidly internalised into the vacuolar system of both cells and protoplasts. However, purified samples of the 70K FITC-dextran were taken up into the vacuoles of cells but not protoplasts after a 1h incubation period. This apparent difference in the ability of cells and protoplasts to internalise FITC-dextrans was confirmed using samples of both commercial and purified 20K FITC-dextran as putative endocytotic probes. Both confocal and conventional fluorescence microscopy of FITC-treated cells have shown that FITC was internalised into similar intracellular compartments as was observed in cells treated with three-times purified 70K FITC-dextran. Thus, FITC was a useful fluorophore for rapidly labelling both the putative endocytotic compartments and the pleiomorphic vacuolar system of carrot cells. Kinetic studies indicated that FITC entered the cell by diffusion in the form of the neutral molecule. We have shown that treatment of cells or protoplasts with the drug Probenecid reversibly inhibited the uptake of FITC from the cytoplasm into the vacuole. In addition, the uptake of FITC into isolated vacuoles was enhanced in the presence of Mg-ATP.
Relationship between preprophase band organization, F-actin and the division site in Allium: Fluorescence and morphometric studies on cytochalasin-treated cells
Authors: Mineyuki Y. , Palevitz B. A.
Published: April 2021

The preprophase band (PPB) of microtubules (Mts), which appears in the G2 phase of the cell cycle in higher plants but disappears well before the end of karyokinesis, is implicated in the determination of the division plane because its location marks the site at which the phragmoplast/cell plate will fuse with the parental plasmalemma during cytokinesis. The PPB first appears as a rather wide array, which progressively narrows before or during prophase. Actin-containing microfilaments (Mfs) have recently been reported in the PPB, but the role of these elements in PPB organization and/or function remains unclear. The present study employed fluorescence and pharmacological methods in symmetrically and asymmetrically dividing epidermal cells of Allium to probe this problem. Our results show that PPBs in cells treated with 2–200 μM cytochalasin D (CD) are still transversely aligned but remain two to three times wider than mature bands in control cells. Treatment for 0.5 h at 20 μM is sufficient to make the PPBs abnormally wide. Premitotic nuclear migration in asymmetrically dividing cells is also inhibited by CD, as is the positioning of the mitotic apparatus and the new cell plate. The plate is still transverse, however. Band-like arrays of cortical Mfs become evident in most interphase cells by prophase. The band remains quite wide compared to the final dimensions of the Mt PPB, and clearly encompasses it. Levels of CD as high as 200 μM decrease the number of cells with transverse actin bands, although a majority still retain them. Other F-actin arrays are disrupted by the drug. Thus, while CD does not inhibit the formation of an initial, broad, transverse PPB in most cells, it does prevent the narrowing process that defines the precise division site. The role of actin in this effect is discussed.
Regulation of integrin mobility and cytoskeletal association in normal and RSV-transformed chick embryo fibroblasts
Authors: Horvath Andrea R. , Kellie Stuart
Published: April 2021

The mobility of the integrin receptor in trypsinised chick embryo fibroblasts (CEF) was investigated using the CSAT monoclonal antibody. The binding of CSAT to trypsinised CEF followed by incubation at 37°C resulted in patching and then capping of the receptor. This capping was dependent on cellular metabolism, since agents such as sodium azide or 2-deoxyglucose inhibited the process. Whereas about 95% of unclustered integrin was soluble in Nonidet P40-containing buffers, after capping more than 25% of surface integrin became detergent-insoluble, indicating a physical association with cytoskeletal elements. Thus the crosslinking of integrin via its βsubunit is sufficient, to induce cytoskeletal association. Unusually, the microfilament-disrupting drugs cytochalasins B and D potentiated CSAT-induced capping in terms of both cell number and the conformation of caps on individual cells. Double immunofluorescent staining demonstrated that in cytochalasin-treated cells both F-actin and talin colocalised with surface CSAT-integrin clusters. The co-distribution of these cytoskeletal components with surface integrin was retained in cytoskeletal preparations, although there was no quantitative increase of either talin or vinculin in the cytoskeletons. The cocapping of talin with integrin clusters on CEF could also be observed in the absence of cytochalasins. No differences were found in capping efficiency, talin and actin co-localisation or cytoskeletal association of surface-modulated integrin in Rous sarcoma virus (RSV)-transformed cells compared with untransformed counterparts, although differences in the response to cytochalasins were observed. These results provide novel evidence for a physiologically relevant association of integrin with cytoskeletal components and its regulation by surface configuration. The importance of this regulation, and the influence of cytoskeleton-specific agents and transformation by RSV are discussed.
Transport of L-tyrosine by B16/F10 melanoma cells: the effect of the intracellular content of other amino acids
Authors: Jara J. R. , Martinez-Liarte J. H. , Solano F. , Peñafiel R.
Published: April 2021

The uptake of L-Tyr by B16/F10 malignant melanocytes in culture has been studied. These melanoma cells can either be depleted of amino acids by 1 h preincubation in Hanks’ isotonic medium or preloaded with a specific amino acid by Ih preincubation in the same solution containing 2mM of the amino acid to be preloaded. By means of these pretreatments, it is shown that the rate of L-Tyr uptake is greatly dependent on the content of other amino acids inside the cells. The L-Tyr uptake is higher in cells preloaded with amino acids transported by the L and ASC systems than in cells depleted of amino acids or preloaded with amino acids transported by the A system. It is concluded that L-Tyr is mainly taken up by an exchange mechanism with other amino acids mediated by the LI system, although the ASC system can also participate in the process. In agreement with that, the homo-exchange performed by cells preloaded with unlabelled L-Tyr is more efficient than any other hetero-exchange, although L-Dopa, the product of tyrosine hydroxylation in melanin synthesis, is almost as efficient as L-Tyr. Apart from aromatic amino acids, melanoma cells preloaded with L-Met and L-His also yield a high initial rate of L-Tyr uptake. The results herein suggest that melanoma cells do not have transport systems specific for L-Tyr, even if this amino acid is needed to carry out the differential pathway of this type of cells, melanosynthesis.
A role for preprophase bands of microtubules in maturation of new cell walls, and a general proposal on the function of preprophase band sites in cell division in higher plants
Authors: Mineyuki Y. , Gunning B. E. S.
Published: April 2021

Time-lapse video microscopy of dividing Tradescantia stamen hair cells that are undergoing cytokinesis has revealed that the maturation of the new cell wall is aided by factors at the site where the preprophase band of microtubules forms before mitosis. The wall changes from being fluid and wrinkled before it is inserted into the parental wall at the end of cytokinesis, to being stiff and flat by about 20 min after the time of attachment. This change occurs only if the new wall is inserted at the site formerly occupied by the preprophase band. The cell plate does not flatten when it is caused to insert elsewhere by drug treatments or by centrifugal displacement. If insertion at the correct site is delayed locally by centrifugation against the direction of expansion of the cell plate, then flattening is delayed at the same locality. In combination with a number of points from the literature of plant cell division, some of them very long-standing, our observations lead to a general proposal regarding the nature of the preprophase band site, its mode of action and timing of its operations, and how its role in spatial regulation of histogenesis is achieved.
Optimization of culture conditions for high cell density proliferation of hl-60 human promyelocytic leukemia cells
Authors: Schumpp Bénédicte , Schlaeger Ernst-Jütrgen
Published: April 2021

The purpose of the present investigation was to optimize the culture conditions in suspension of the HL-60 cell line for high-density production. The optimized HL medium was a mixture of RPMI-1640, DMEM, HamF12 and IMDM media supplemented with transferrin, insulin, Primatone RL, Pluronic F68, ethanolamine and selenite. Under these conditions, whether serum was added or not, cells grew to up to 8 ×108 cells ml−1, which was at least three times higher than the maximum cell density usually described. Glucose and four amino acids: cystine, glutamine, methionine and serine, were highly consumed and disappeared quickly from the medium. Nutrient supply and metabolic end-product accumulation were the most probable growth-limiting factors. Different propagation systems were used to increase the cell density further. Cells were grown in dialysis tubing where relatively high levels of nutrients and low levels of waste products were maintained, leading to cell densities of 60×10® to 70×10® cells ml−1. A perfusion-culturing method with cell retention was found to be most effective for high-density production of HL-60 cells at a 21 as well as a 601 fermenter scale. Average concentrations of 40×106 to 50×106 cells ml−1 were achieved. Expression and distribution of both tumor necrosis factor receptors (55 and 75×103Mr) on the surface of HL-60 cells were analysed as a control of the physiological integrity of the cells during the perfusion course; 1.3μg of tumor necrosis factor receptors/1010 cells was regularly expressed on the surface of HL-60 cells.
Resinless section immunogold electron microscopy of karyo-cytoskeletal frameworks of eukaryotic cells cultured in vitro: Absence of a salt-stable nuclear matrix from mouse plasmacytoma MPC-11 cells
Authors: Wang X. , Traub P.
Published: April 2021

The karyo-cytoskeleton of cells cultured in vitro was investigated employing resinless section immunogold electron microscopy. Cells were entrapped in low-melting agarose, sequentially extracted with various buffers and digested with nucleases to obtain karyo-cytoskeletal frameworks and reacted with specific primary and gold-conjugated secondary antibodies or gold-conjugated protein A to decorate structural elements of these frameworks. Following embedment of the gold-labeled residual cell structures in diethylene glycol distearate and their sectioning, the embedding material was removed with organic solvent and the sections were finally subjected to CO2 critical point drying. When this technique was applied to mouse skin fibroblasts (MSF), it revealed a dense and salt-stable intranuclear network of fibrogranular material. Antibodies directed against vimentin and lamin B detected a cytoplasmic meshwork of intermediate filaments (IFs) and a nuclear lamina, respectively; the latter, however, only after removal of chromatin from nuclei by nuclease digestion of DNA. Intranuclear filaments free of adhering globular material were morphologically very similar to cytoplasmic vimentin filaments. By contrast, mouse plasmacytoma MPC-11 cells lacking detectable amounts of cytoplasmic IF proteins and lamins A and C were devoid of a salt-stable internal nuclear matrix. The same holds true for MPC-11 cells that had been treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate to induce vimentin synthesis and establish a cytoplasmically extended IF network. These findings were in accordance with the biochemical behavior of Triton X-100-treated MSF and MPC-11 cells and their appearance in immunofluorescence microscopy upon extraction with high ionic strength buffer. While the chromatin was quantitatively retained in the residual cell structures derived from MSF cells, in those obtained from MPC-11 cells the nuclear lamina was disrupted and the chromatin was released from the nuclei, suggesting that MPC-11 cells lack the salt-stable nuclear scaffold to which chromatin is normally anchored.
Architecture of the outer arm dynein ATPase in an avian sperm flagellum, with further evidence for the B-link
Authors: Burgess S. A. , Dover S. D. , Woolley D. M.
Published: April 2021

Demembranated sperm flagella from Gallus domesticas have been prepared by the rapid-freeze, deepetch, rotary replica technique in order to study the three-dimensional morphology of the outer dynein arm (ODA) –ATPase complex. In general, the ODAs resemble most closely those from the sea urchin Strongylocentrotus described by W. S. Sale et al. In the ‘rigor’ condition, the ODA consists of a mqjor subunit (the head), from which a slender link extends to the adjacent B-tubule (the B-link). A smaller, intermediate subunit lies adjacent to the head, distally, and a further extension of the complex, the minor subunit, continues distally beneath the head domain of the next arm complex, where it attaches to the A-tubule. In the presence of ATP and vanadate (‘relaxed’ condition), the attachment point of the B-link to the head is shifted to a more proximal position, and the minor subunit is no longer visible. This is interpreted as resulting from a rotation of the head. These features are demonstrated stereoscopically, and from several viewpoints. Image enhancement has been used to clarify and define the repetitive features of the dynein arrays. In addition, some of the axonemes have been imaged from highly contrasted 20 nm thin sections; the detection of B-links in such sections means that these slender structures cannot be considered artefacts of the rapid-freeze, deep-etch protocol.