bluuebunny/crossref_metadata_2025 huggingface dataset:
Oligodendrocytes repel axons and cause axonal growth cone collapse
Authors: Fawcett J. W. , Rokos J. , Bakst I.
Published: April 2021

We have examined the interactions between axons regenerating from dorsal root ganglia (DRGs) derived from newborn rats and oligodendrocytes cultured by three different techniques. Cultures examined after 2 days have a profuse outgrowth of axons from the DRGs, forming a dense mat on the culture surface. However, the axons avoid growing on oligodendrocytes; axons are seen all around these cells, but do not grow over them. We have also performed time-lapse video studies of the interactions between axonal growth cones and oligodendrocytes. Axons grow normally until their growth cone comes into direct contact with an oligodendrocyte, following which the growth cone remains motile for 30-60 min, but without making any progress over the cell. The growth cone then suddenly collapses, and the axon retracts, leaving a thin strand in contact with the cell. After this a new growth cone is usually elaborated and the process repeated. Oligodendrocytes are therefore inhibitory to axonal growth, and this may partially explain the failure of axons to regenerate in the mammalian central nervous system.
Glycophorin expression in murine erythroleukaemia cells
Authors: Ulmer Jeffrey B. , Dolci Elizabeth D. , Palade George E.
Published: April 2021

We have identified mature and putative precursor forms of glycophorins expressed in a virus-transformed murine erythroleukaemia (MEL) cell line and compared them with their normal erythroblast counterparts. The following differences were found: (1) the two major MEL cell glycophorins (apparent Mr values 29–30 and 43(×103)) have greater mobility on polyacrylamide gels than their normal gp-3 and gp-2 counterparts, due at least in part to differences in their oligosaccharide sidechains; (2) MEL cell gp-3 consists of two discrete proteins; and (3) there are more potential glycophorin precursors in MEL cells than in normal mouse erythroblasts. Four proteins, with apparent Mr values of 21, 23, 26 and 27(×103), have tentatively been identified as glycophorin precursors, based on the following findings: (1) they are immunologically related to the glycophorins; and (2) their synthesis was induced by dimethyl sulphoxide coincidentally with that of gp-3 and gp-2. They do not appear to be glycoproteins, as evidenced by their lack of incorporation of [3H]galactose, [3H]glucosamine or [3H]mannose. In contrast, gp-3 and gp-2 incorporated [3H]galactose and [3H]glucosamine but not [3H]mannose. Partial characterization of the glycan moieties of MEL cell glycophorins indicates that they consist mostly of tri- and tetrasaccharides, with no indication of any N-linked chains. Hence, the glycans of MEL cell glycophorins are mostly (if not all) O-linked. Furthermore, treatment with N-glycanase did not change their electrophoretic mobility on polyacrylamide gels. MEL cell glycophorins were also shown to be modified by phosphoryl and fatty acyl groups.
Coated vesicles from chicken liver bind ferritin
Authors: Passaniti Antonino , Roth Thomas F.
Published: April 2021

The iron-storage protein ferritin was found to be associated with highly purified coated vesicles (CV) from chicken liver. Chicken liver ferritin was morphologically similar to ferritin from horse spleen and could be isolated using a specific anti-ferritin monoclonal antibody. This antibody recognized a 240 ×103Mr form of chicken ferritin but not the 22 ×103Mr ferritin subunit after protein transfer to nitrocellulose. CV purified by controlled-pore glass-bead chromatography also contained ferritin when assayed by monoclonal anti-ferritin antibody using a sensitive enzyme-linked assay. Ferritin remained associated with CV even after re-chromatography. Ferritin particles were observed to be associated with CV by electron microscopy. CV-associated ferritin could be quantitatively removed from CV by treatment of the CV with 0 · 5M-Tris-HCl + 2 M-urea at pH 8 · 5, conditions that also lead to dissociation of the clathrin lattice. Triton X-100 detergent treatment did not affect the association of ferritin with CV. These results indicate that purified CV from chicken liver contain ferritin in association with the clathrin lattice. The possible functional significance of this association is discussed.
Uneven distribution of surface antigens during antigenic variation in Paramecium primaurelia
Authors: Antony Claude , Capdeville Yvonne
Published: April 2021

In Paramecium primaurelia surface antigen (SAg) expression can be experimentally controlled by temperature-shift-induced antigenic variation. As only one SAg is usually expressed at the cell surface under stable environmental conditions, we used the temperature-shift-induced change in SAg to follow the newly expressed antigen and the disappearing one, by both immunofluorescence and immunogold electron microscopy. The new SAg initially appeared scattered at the cell surface, over the ciliary and interciliary membrane domains, without any readily identifiable specific site of insertion into the plasma membrane. The concentration of the newly incorporated molecules then increased gradually on the plasma membrane. In contrast, the surface distribution of the previously expressed SAg was not complementary to the pattern of the appearing SAg. The loss of the old SAg is delayed after the temperature shift and seems to occur more suddenly than the appearance of new SAg. This loss is characterized by a subpopulation of cilia bearing old SAg coexisting with other cilia and a pellicle almost devoid of the old SAg molecules. The topological distribution of the new and old SAgs is discussed in relation to the lipidie nature of the SAg membrane anchor and to a possible role of an endogenous Paramecium phosphatidylinositol phospholipase C.
A serum factor inducing neurite retraction of morphologically differentiated neuroblastoma × glioma NG108-15 cells
Authors: Ghahary A. , McPherson P. S. , Cheng K. W.
Published: April 2021

It is well established that serum plays an important role in cell proliferation and differentiation. In this study, we have identified a serum factor that induces rapid neurite retraction of morphologically differentiated NG108-15 cells, cultured in serum-free medium containing 1 mM-dibutyryl cyclic AMP. The serum fraction of Mr > 30×103 induces neurite retraction in a manner identical to that of the whole serum. The neurite retraction activity in serum appears to be acid- and heat-stable. The molecular weight of the serum neurite retraction factor (NRF) has been demonstrated to be approximately 70×103 by gel permeation on LKB-Ultrogel AcA-44. The neurite retraction activity is dosedependent, and the time required for half-maximal activity (t2) is 1-8 min. NRF is present in sera of various species studied, including human, cattle, sheep, rabbit and horse, but not in tissue extracts of kidney, heart, lung, skeletal muscle, and brain of the rat. However, rat spleen and liver homogenates, at a protein content of 1 mg ml−1, caused slight neurite retraction. It is noteworthy that NRF is not detectable in cerebral spinal fluid. Our data on the properties of serum NRF indicate that it differs from all of the well-established growth factors, namely, NGF, EGF, PDGF, FGF, NSILA, ECGF and TGF. Further studies on purified NRF will delineate the biological role(s) of this serum factor in the process of maturation and differentiation of developing neurones.
Glycosaminoglycans facilitate the movement of fibroblasts through three-dimensional collagen matrices
Authors: Docherty R. , Forrester J. V. , Lackie J. M. , Gregory D. W.
Published: April 2021

The effect of glycosaminoglycans on the invasion of choroid fibroblasts into type I collagen gels was studied. Both hyaluronate and chondroitin sulphate, when incorporated into the gel, facilitated invasion of the collagen matrix, although hyaluronate was considerably more effective. Hyaluronate-induced fibroblast invasion was markedly concentration-dependent, being reduced at both high and low concentrations. Increased cell invasion appeared to correlate with denser packing of collagen fibrils within the gel, since the same effect could be achieved by increasing the collagen concentration of native, i.e. glycosaminoglycan-free gels. Scanning electron microscopy of the interior of the collagen gels suggested that changes in packing arrangement of fibrils in gels that had polymerized in the presence of glycosaminoglycans might account in part for different rates of cell invasion.
Kinetochore size variation in mammalian chromosomes: An image analysis study with evolutionary implications
Authors: Cherry Lorraine M. , Faulkner Alexander J. , Grossberg Leigh Ann , Balczon Ronald
Published: April 2021

The kinetochore, a proteinaceous plate that is the site for attachment of spindle microtubules to the metaphase chromosome, can be visualized using anti-kinetochore indirect immunofluorescence. We have used computer-assisted image analysis to measure the variation of kinetochore surface areas, as reflected by immunofluorescence areas, in cell lines derived from rat kangaroo, Chinese hamster and common rat, to determine if our size estimates correlate well with those obtained using measurements from electron micrographs. In addition, we used male and female human fibroblast cell lines, as well as a transformed human female cell line (HeLa), to examine kinetochore size variation among cells, between sexes, and between cell lines. We found that our system gave reproducible estimates of kinetochore size, and that these sizes correlated very well (r=0 ·95) with the electron micrograph measurements. In examining variation within humans, we observed measurable differences between cell lines. Despite this difference, all the human lines had size distributions that were leptokurtotic and positively skewed. The fact that very few chromosomes exhibited areas smaller than the mode gives support to the idea that mammalian chromosomes may require a specific, minimum amount of kinetochore material in order to maintain stable attachment to the mitotic spindle. On the other hand, the positive skewness seems to indicate that larger kinetochores, possibly the result of events such as Robertsonian fusions, are fully functional. The retention of this plasticity may allow the chromosomes to maintain an evolutionary adaptability that might otherwise be lost.
The transient division block in the D9ts mutant of Tetrahymena is inducible at every cell cycle stage
Authors: Cleffmann Günter
Published: April 2021

The temperature-sensitive mutant D9 of Tetrahymena thermophila doubles its size at restrictive temperature. It does so by complete cessation of cell division for a limited time. After resumption of proliferation, division rate and specific growth rate are the same as at the permissive temperature, thereby maintaining the new cell size. In this study a detailed analysis of the process of controlling the new cell size is presented, by probing the temperature sensitivity of cell cycle phases. It will be shown that high temperature affects the size-controlling system immediately upon shift in temperature. Temperature pulses are effective at every stage in the cycle and are executed at the time of expected division. After return to the permissive temperature, cells gradually recover from the temperature pulse as seen by a decrease in division delay. Preparation for the next division is unaffected by the temperature pulse. It occurs at the same time as in untreated controls. The results allow us to describe some features of the division initiating system.
A monoclonal antibody raised against cytoplasmic fibrillar bundles from carrot cells, and its cross-reaction with animal intermediate filaments
Authors: Hargreaves Alan J. , Dawson Peter J. , Butcher Geoffrey W. , Larkins Audrey , Goodbody Kim C. , Lloyd Clive W.
Published: April 2021

Carrot suspension cells contain cytoplasmic bundles of fibrils that are distinct from F-actin and microtubules and have some of the characteristics of intermediate filaments. In characterizing these fibrillar bundles further, we have raised a monoclonal antibody against them. This anti-fibrillar bundle antibody (AFB) immunoblots vimentin from a range of animal cells and tissues, as well as glial fibrillary acidic protein in brain and desmin in BHK fibroblasts, which are representatives of the type III intermediate filaments.

Immunofluorescence staining of PtK2 cells indicates that AFB recognizes a network co-distributing with cytoplasmic microtubules. When this co-alignment is disturbed with the anti-microtubule agent, colcemid, the AFB staining segregates with the collapsed perinuclear whorls of vimentin.

In carrot, AFB immunoblots the major bundle proteins but not plant tubulin. In plant as in animal cells, AFB immunofluorescently labels antigens that co-distribute with microtubules. In onion cells (which, unlike carrot, do not possess paracrystalline arrays of fibrils) AFB labels all four microtubule arrays throughout the cell cycle. The antigens do not, however, collapse around the spindle poles during mitosis.

Double immunofluorescence, using anti-dog brain tubulin, indicates that the FB antigen is more diffusely distributed than tubulin; it is patchy and co-alignment is not exact, particularly during early preprophase band formation.

Antigens in detergent-insoluble fibrils of carrot cells therefore exist both in animal type III intermediate filaments (IF), and in a more dispersed, microtubule-associated manner in onion meristematic cells. This constitutes an independent line of evidence for the existence of IF antigens in plants.
Retention of ciliary ninefold structure after removal of microtubules
Authors: Stephens R. E. , Oleszko-Szuts S. , Linck R. W.
Published: April 2021

When axonemes of isolated gill cilia from the bay scallop Aequipecten irradians are heated at 45°C for a minimum of 8 min in a 10mM-Tris-HCl (pH 8), ImM-EDTA solution, nearly 80% of the tubulin is solubilized but most minor structural proteins are retained in a ninefold symmetrical configuration. This remnant consists of the junctional protofilaments, derived from outer doublet tubules, interconnected by nexin linkages, with radial spoke components still directed inwards. The remnant is of the same length as the original cilium, with the junctional protofilaments attached at the distal end to the ciliary tip and at the proximal end to the basal plate. Virtually identical fractionations can be achieved with blastula cilia isolated from both arctic and tropical sea-urchin embryos. The remnant is resistant to salt up to at least 1M concentration, judged by the constancy of protein composition. Immunoblotting with antibodies against sea-urchin sperm flagellar tektins indicates that the tektins remain within the ciliary remnant, supporting their location within the junctional protofilament domain. The fractionation is inhibited by low pH, by magnesium or calcium ions in the millimolar range, and by monovalent ions at 10-fold higher concentrations. About a quarter of the total ciliary calmodulin is bound to the axoneme at micromolar calcium levels but most is released upon thermal fractionation. Polymerization of tubulin in the presence of the remnant results in singlet microtubules, separate from the remnant proper, suggesting that doublet formation may require coordinate co-assembly of tubulin with skeletal proteins. These observations demonstrate the existence of a fibrous skeleton in the axoneme, composed largely of ciliary tektins, nexin linkages, and other structural proteins.
Properties of microtubule-free cortical residues isolated fromParamecium tetraurelia
Authors: Williams Norman E. , Honts Jerry E. , Stuart Kathleen R.
Published: April 2021

We have found that shape-preserving residues devoid of microtubules can be prepared from Paramecium using Triton X-100 at high ionic strength. These residues contain many proteins, including one showing antigenic relationship to chicken a-spectrin, and three showing antigenic relationship to Tetrahymena cortical proteins. These antigens have been localized by immunofluorescence microscopy, and the isolated cortical residues have been characterized ultrastructurally. These preparations should be useful in detailed studies of the structure, development and evolutionary relationships of cortical architecture in ciliated protozoa.
Identification and localization of major cortical proteins in the ciliated protozoan, Euplotes eurystomus
Authors: Williams Norman E. , Honts Jerry E. , Lu Qun , Olson Cheryl L. , Moore Kenneth C.
Published: April 2021

Shape-preserving cortical residues have been isolated from Euplotes eurystomus cells by the application of Triton X-100 at high ionic strength. These integrated structures consist of articulated plates and widely interspersed cages that formerly contained basal bodies associated with the clusters of cilia characteristic of this cell type. Using SDS-PAGE and immunolocalization procedures, we have identified major subunit proteins of both the plates (116, 110 (×103)Mr, and the basal body cages (86× 103Mr). The potential for studies of these proteins in contributing to our understanding of cortical development and evolution in Euplotes is discussed.
Effect of hypoxia upon the growth and sprouting activity of cultured aortic endothelium from the rat
Authors: Smith Paul
Published: April 2021

Endothelial cells were obtained from the aortas of Wistar rats by collagenase digestion. Cells were grown to confluence in medium 199 enriched with L-glutamine but without specific growth factors. Cells were subcultured into 35 mm dishes or 25 cm2 flasks coated with fibronectin. For cell growth studies, cells were seeded onto multiwell plates or 35 mm dishes. In two experiments the cells were grown in an hypoxic atmosphere of 5·3% O2 and in a third the level of oxygen was 2·5%. Control cultures for each experiment were grown in 5% CO2 and air. Cell populations were counted at 2-day intervals and at the termination of each experiment the cells were fixed and the area of each plate or flask occupied by sprouting cells was assessed by point counting.

Endothelial cells grown in 5·3% O2 grew more rapidly and attained confluence earlier than in the controls. An atmosphere of 2·5% O2 did not accelerate growth but neither did it inhibit it, so after 9 days there were as many hypoxic cells as there were controls. Hypoxia also stimulated sprouting activity to occur earlier and to become much more extensive than in control cultures. Under the influence of hypoxia, sprouting consisted of complex anastomotic or arborizing patterns forming syncytium-like masses beneath the monolayer of oval cells. This process appeared to originate from foci of altered endothelial cells that had become retracted, smaller, elongated and migratory, and which displayed increased immunoreactivity for factor VIII antigen. It was concluded that a level of hypoxia, similar to that in systemic veins, stimulates growth of arterial endothelium and provokes enhanced sprouting activity. This behaviour may be relevant to the process of vascularization of wounds or neoplasms.
Differences in the exposure of c- and n-terminal tubulin domains in cytoplasmic microtubules detected with domain-specific monoclonal antibodies
Authors: Dráber Pavel , Dráberová Eduarda , Linhartová Irena , Viklický Vladimír
Published: April 2021

A panel of 11 monoclonal antibodies specific to α- or β-tubulin subunits was used to study the location of tubulin molecules in cytoplasmic microtubules. Specificity of antibodies was confirmed by immunoblotting and immunofluorescence experiments on fixed cells. The limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of both subunits. Epitope mapping of isolated a-tubulin revealed that a set of antibodies against the N-terminal domain of the α-subunit (TU-01, TU-02, TU-03, TU-09, 6-11B-1) recognized at least four different antigenic determinants. Immunofluorescence staining of unfixed detergent-extracted cells showed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies to N-terminal domains. The same results were obtained after microinjection of antibodies into living cells. The unchanged distribution of microtubules in injected cells was confirmed by double-label immunofluorescence with polyclonal antibodies. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of the microtubules, considerable regions of the N-terminal domains are either not exposed on the surface of cytoplasmic microtubules, or are masked by interacting proteins.
NACM, A cytopathogen from naegleria ameba: purification, production of monoclonal antibody, and immunoreactive material in NACM-treated vertebrate cell cultures
Authors: Dunnebacke T. H. , Dixon J. S.
Published: April 2021

An amebic component NACM (Naegleria ameba cytopathogenic material) is obtained from free-living amebae and acts as a cytopathogen in cultured avian and mammalian cells. NACM has been purified and partially characterized using a scheme that includes ammonium sulfate precipitation, liquid chromatographic separations and electrophoretic isolations. The purified NACM product is biologically active. Its properties, revealed by its behavior following treatment with enzymes and during purification, are those of a protein; its size is in the molecular weight range of 36 000 and it has an isoelectric point of pH 4·2. Monoclonal antibodies have been produced to NACM that prevent its cytopathic activity, and, in dot-blot procedures, identify purified NACM-containing fractions. When used as immunostains, NACM antibodies disclose the presence of immunospecific material both in the amebae and in vertebrate cells inoculated with NACM. In the amebae, the immunostain is located at the tips of the pseudopodia and in the peripheral cytoplasm; in the vertebrate cells, it is absent in uninoculated cells and is not observed in the inoculated cells for the first 3–4 days after the addition of the NACM to the culture. Subsequently, in a time course associated with the development of cytopathic changes observed in the light microscope, immunostaining material develops in the perinuclear cytoplasm where it condenses into a large mass before the cells undergo lysis. On the basis of these results, it appears that NACM represents an unprecedented system in which a protein material from an organism, in the course of destroying unrelated cells, causes the production of a cytoplasmic product that is immunologically recognizable as the inoculating amebic material.
Studies on the biochemistry and morphology of freshly isolated and maintained rat hair follicles
Authors: Philpott Michael , Green Martin R. , Kealey Terence
Published: April 2021

We have already shown that shearing can be used to yield large numbers of viable intact hair follicles. We now show that these follicles can be viably maintained on permeable supports for 7 days in vitro as determined by their adenine nucleotide contents, rates of [mefhyl-3Hlthymidine and [U-14C]leucine uptake, [methyl-H]thymidine autoradiography, patterns of keratin synthesis and light and electron microscopy. These studies, however, show that after 7 days maintenance the morphology of maintained follicles shows a closer resemblance to the telogen rather than the anagen follicle. We therefore conclude that the failure of previous attempts at maintaining hair growth in culture is due to hair follicles prematurely entering the resting stage of their hair growth cycle, possibly as a response to isolation.
On the distribution of microtubule-associated intermediate filament antigens in plant suspension cells
Authors: Goodbody Kim C. , Hargreaves Alan J. , Lloyd Clive W.
Published: April 2021

Intermediate filament antigens are known to coalign, patchily, with cortical microtubules in plant cells, but nothing else is established about this relationship or the form the antigens take. This was studied further using cell suspensions, instead of root tip cells, since their cortex is accessible to antibodies without the rigours of cell separation. Both carrot and sweetcorn suspension cells were labelled with three antibodies known to recognize animal intermediate filaments. The mitotic and cytokinetic apparatus could be stained with these antibodies, which, in interphase cells, also labelled cortical microtubule-like arrays. One antibody (AFB), raised against cytoplasmic bundles of 7nm fibrils from carrot cells, immunostained the bundles but only under conditions of fixation that did not allow the finer, microtubule-associated staining pattern to be seen. By exploring various preparatory conditions it was concluded that these two forms of antigen co-exist: they are not experimentally interconvertible but require different conditions for exposure to different antibodies.

Double immunofluorescence established that the intermediate filament antigens do not parallel the actin network, nor did cytochalasin D affect their distribution. Taxol, however, bundled the intermediate filament antigens, whereas they are dispersed when microtubules are depolymerized, rather than collapsing in perinuclear whorls.

Under conditions permitting the microtubule-associated antigens to be stained by immunofluorescence, carrot protoplasts were cleaved on grids, exposing the cortical microtubules. Immunogold labelling then showed that the antibody raised against fibrillar bundles recognizes patches of electron-dense material, along and between the microtubules, rather than individual filaments. To confirm that the plant antigens are capable of forming filaments, a high salt, detergent-insoluble fraction was prepared from the maize line. By dialysing from urea, intermediate-sized filaments could be reconstituted and they immunoblotted with the broadly cross-reactive antibody to intermediate filament antigens (anti-IFA).

These studies underline the problems of visualizing in plants, conformations of intermediate filament antigens that are not directly comparable to the extensively studied animal models, and that appear to be sensitive to the way in which cells are manipulated.
Kinetochore microtubules shorten by loss of subunits at the kinetochores of prometaphase chromosomes
Authors: Cassimeris Lynne , Salmon E. D.
Published: April 2021

The site of tubulin subunit dissociation was determined during poleward chromosome movement in prometaphase newt lung cell mitotic spindles using fluorescence photobleaching techniques and nocodazole-induced spindle shortening. Synchronous shortening of all kinetochore microtubules was produced by incubating cells in 17 μM nocodazole to block microtubule assembly. Under these conditions the spindle poles moved towards the metaphase plate at a rate of 3.6±0.4 μmmin−1 (n=3). On the basis of anti-tubulin immunofluourescent staining of cells fixed after incubation in nocodazole, we found that nonkinetochore microtubules rapidly disappeared and only kinetochore fibers were present after 60–9 0 s in nocodazole.

To localize the site of tubulin subunit dissociation, a narrow bar pattern was photobleached across one half-spindle in prometaphase-metaphase cells previously microinjected with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF)-labeled tubulin. Immediately after photobleaching, cells were perfused with 17 μM nocodazole to produce shortening of kinetochore microtubules. Shortening was accompanied by a decrease in the distance between the bleach bar and the kinetochores. In contrast, there was little or no decrease in the distance between the bleach bar and the pole. Compared to their initial lengths, the average kinetochore to pole distance shortened by 18 %, the bleach bar to kinetochore distance shortened by 28% and the average bleached bar to pole distance shortened by 1.6%. The data provide evidence that tubulin subunits dissociate from kinetochore microtubules at a site near the kinetochore during poleward chromosome movement These results are consistent with models of poleward force generation for chromosome movement in which prometaphase-metaphase poleward force is generated in association with the kinetochore.
Neomycin reversibly disrupts mitotic progression in stamen hair cells of Tradescantia
Authors: Larsen Paul M. , Chen Tung-Ling L. , Wolniak Stephen M.
Published: April 2021

Neomycin has been reported to inhibit polyphosphoinositide cycling by preventing the hydrolysis of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Inositol 1,4,5-trisphosphate, through the mobilization of calcium, and 1,2-diacylglycerol, through the activation of protein kinase C, trigger many physiological responses. The addition of 2 mM neomycin to stamen hair cells of Tradescantia virginiana at various [mints during mitosis arrests cells in prophase, prior to nuclear envelope breakdown, or in metaphase. Arrest in prophase is irreversible. Metaphase arrest can persist for over 2h before the cells attempt to revert to interphase without dividing. Entry into anaphase by the majority of cells in our sample arrested in metaphse occurred after treatment with 1,2-dioctanoylglycerol while 1,3-dioctanoylglycerol was totally ineffective at reversal. Perfusion of 100 μM calcium chloride solution past the cells was sufficient to reverse arrest in approximately half of the cells in the sample. Magnesium could not be substituted for calcium in the reversal. Clindamycin, another member of this class of aminoglycoside antibiotics, with no known inhibitory effect on polyphosphoinositide cycling, is without effect on mitotic progression in stamen hair cells. Our results indirectly implicate one or more episodes of polyphosphoinositide cycling and its resultant protein phosphorylation by protein kinase C in the regulatory cascade that leads to anaphase.
Polarity in spontaneous unwinding after prior rotation of the flagellar beat plane in sea-urchin spermatozoa
Authors: Takahashi Keiichi , Shingyoji Chikako , Katada Jun , Eshel Dan , Gibbons I. R.
Published: April 2021

The flagellar beat plane of live and reactivated seaurchin sperm held by their heads in the tip of a vibrating micropipette will rotate along with the plane of the imposed vibration for up to 10 revolutions in either a clockwise or a counterclockwise direction. Subsequent cessation of the imposed vibration is followed by spontaneous unwinding of the flagellar beat plane. Nearly complete unwinding occurs after prior counterclockwise winding. The unwinding of the beat plane after prior clockwise winding is incomplete, but the number of revolutions that remain unwound affects the response of the flagellar beat plane to a second set of imposed revolutions. The initial angular velocity of spontaneous unwinding is approximately proportional to the number of prior winding cycles, independent oftheir direction. The maximum initial velocity of unwinding was 27rad s−1 and 20rad s−1 for live and reactivated sperm, respectively. These data suggest that the force responsible for unwinding of the beat plane is derived from the elastic distortion of some component in the axonemal structure. The difference in completeness of spontaneous unwinding between the two directions of rotation is consistent with the previously suggested hypothesis that imposed rotation of the beat plane reflects the forced rotation of the central pair within the axoneme.
Abnormal chromatin (abc), a maternal-effect locus in Drosophila melanogaster
Authors: Vessey Kristin B. , Ludwiczak Rachael L. , Briot Amy S. , Underwood Eileen M.
Published: April 2021

Mutations in the maternal-effect gene abnormal chromatin (abc) in Drosophila melanogaster result in a variety of defects involving nuclear replication/ division. Three recessive alleles of this gene, which maps near 51F on chromosome 2, all result in female sterility. They cause slower embryonic development that is usually abnormal from the earliest nuclear divisions and arrested by the sixth one. Nuclei tend to be large and erratically distributed, some intensely staining. Mitotic asynchrony is common. Few embryos reach the gastrula stage and none hatch. With the weakest allele, fsPL, bridges between nuclei are common; abnormal chromatin clumps that resemble yolk nuclei occur before the other nuclei reach the surface; and spindle anomalies and DNA wads with numerous centrosomes are seen. Females with the stronger alleles, fsA5 and fs27, lay fewer eggs and a smaller proportion of embryos reach blastoderm; developmental arrest occurs earlier, usually with several large nuclei distributed along the length of the embryo. Chorion defects occur in all three mutants. Mitotic asynchrony, nuclear bridging, endoreduplication and nuclear behavior aberrant from the first division suggest that the abc gene product operates in DNA replication/nuclear division. Larval (homozygous F1) neuroblast chromosome structure and mitotic indices are normal, indicating that any mitotic function is strictly maternal, i.e. abc is not a general mitotic gene. Thus abc is one of a few known genes with a maternal effect that appears to function in the embryonic cell cycle.
Implications of DNA replication for eukaryotic gene expression
Authors: Wolffe Alan P.
Published: April 2021

DNA replication has a key role in many developmental processes. Recent progress in understanding events at the replication fork suggests mechanisms for both establishing and maintaining programs of eukaryotic gene activity. In this review, I discuss the consequences of replication fork passage for pre existing chromatin structures and describe how the mechanism of nucleosome assembly at the replication fork may facilitate the formation of either transcriptionally active or repressed chromatin.
Cytoskeletal targets for oncogenic tyrosine kinases
Authors: Kellie S. , Horvath A. R. , Elmore M. A.
Published: April 2021

The past decade has seen intense investigation into the role of tyrosine phosphorylation in cell growth and tumorigenesis (Cooper and Hunter, 1983). Whilst it is now well established that receptor occupancy by a number of growth factors is associated with tyrosine phosphorylation, the signal transduction pathways and the mechanism by which oncogenic tyrosine kinases induce cell growth and neoplastic changes remain elusive (Jove and Hanafusa, 1987; Yarden and Ullrich, 1988). The acutely transforming avian sarcoma viruses have been invaluable tools for examining the molecular basis underlying the changes in cell morphology and growth regulation that occur during neoplasia. The src gene product of Rous sarcoma virus (RSV) is probably the best-characterised oncogenic tyrosine kinase, and we shall review the evidence showing that components of the cytoskeleton are functional targets for this enzyme.
Assembly of a chondrocyte-like pericellular matrix on non-chondrogenic cells Role of the cell surface hyaluronan receptors in the assembly of a pericellular matrix
Authors: Knudson Warren , Knudson Cheryl B.
Published: April 2021

In this study, we have examined the capacity of various cell types, which express cell surface hyaluronan receptors, to organize a chondrocyte-like pericellular matrix when given chondrocyte-derived extracellular matrix macromolecules exogenously. The assembly of a pericellular matrix was visualized by a particle exclusion assay. Without the addition of exogenous macromolecular components, none of the cell types studied exhibited significant pericellular matrices extending from their plasma membranes. However, upon the addition of high molecular weight hyaluronan in combination with aggregating cartilage proteoglycan monomers, large pericellular matrices were formed within two hours of incubation. No pericellular matrices were formed if these macromolecular components were added separately at equivalent concentrations or if the components were added in the presence of hyaluronan hexasaccharide, a competitive inhibitor of hyaluronan interaction with cell surface hyaluronan receptors. Fully assembled pericellular matrices could also be displaced by the subsequent addition of hyaluronan hexasaccharides. Nonliving, glutaraldehyde-fixed cells, which retained functional hyaluronan receptors, maintained the capacity to assembly pericellular matrices with exogenous components, in serumcontaining or serum-free medium. Cells that were incubated with exogenous matrix macromolecules for 24 h, followed by a chase incubation in medium minus the exogenous macromolecules, continued to maintain the matrix for up to 6 h on live cells and more than 24 h on glutaraldehyde-fixed cells. Cell types that did not express hyaluronan receptors were not capable of organizing such pericellular matrices when incubated with these exogenous components. These findings suggest that cells expressing hyaluronan receptors have a significant capacity to organize their immediate extracellular environment via hyaluronan-hyaluronan receptor interactions. Possible physiological functions for this type of matrix organizing capacity are discussed.
Effects of β-xylosides on proteoglycan biosynthesis and morphology of PC12 pheochromocytoma cells and primary cultures of rat cerebellum
Authors: Margolis R. K. , Goossen B. , Tekotte H. , Hilgenberg L. , Margolis R. U.
Published: April 2021

We have examined the effects of β-xylosides, which act as exogenous acceptors for glycosaminoglycan chain initiation, on the morphology and proteoglycan biosynthesis of PC 12 pheochromocytoma cells, and on monolayer, aggregate and explant cultures of early postnatal rat cerebellum. PC 12 cells cultured for 13 days in the presence of nerve growth factor (NGF) and β-xyloside, and labeled during days 11-13 with sodium [35S]sulfate, showed an 8- to 11-fold increase in [35S]sulfate-labeled macromolecules released into the culture medium. Most of the increase was accounted for by chondroitin sulfate, which was in the form of free glycosaminoglycan chains, which were not acid-precipitable. The presence of β-xylo-side also led to a 65-115% increase in [35S]sulfate incorporation into cell-associated glycosaminoglycans and glycoproteins of untreated and NGF-treated PC 12 cells, respectively. β-Xyloside treatment reduced the size of the chondroitin sulfate chains in both the cells and medium from approximately 34 000 to 10000Mr, but had much less effect on heparan sulfate, which decreased in size from 16000 to 13 000-14 500 Mr (in the medium and cells, respectively). β-Xyloside inhibition of proteoglycan biosynthesis was accompanied by significant morphological effects in NGF-treated PC12 cells, consisting of an increase in length and decrease in the branching, diameter and adhesion to the collagen substratum of the PC 12 cell processes. p-Nitrophenyl- and 4-methylumbelliferyl-β-D-xylosides produced similar effects, which were not seen with p-nitrophenyl-β-D-galactoside. fi-Xylosides also produced distinct alterations in the adhesion and morphology of monolayer, aggregate, and explant cultures of early postnatal rat cerebellum, which occurred together with inhibition of chondroitin sulfate proteoglycan biosynthesis and a decrease in glycosaminoglycan chain size. These studies indicate that chondroitin sulfate (and probably also heparan sulfate) proteoglycans play a significant role in modulating cell-cell and cellmatrix interactions in nervous tissue development and differentiation.
Cytoskeletal rearrangements accompanying Salmonella entry into epithelial cells
Authors: Finlay B. Brett , Ruschkowski Sharon , Dedhar Shoukat
Published: April 2021

Salmonella bacteria can enter (invade) eukaryotic cells, and exist as intracellular parasites. Confocal, light immunofluoresence and electron microscopy were used to examine various cytoskeletal components of cultured Madin Darby canine kidney (MDCK) and HeLa epithelial cells after infection with Salmonella typhimurium. These bacteria entered and remained within membrane-bound vacuoles and were surrounded by large (5-10 μm) dense structures composed of various cytoskeletal components. These structures consisted of extensive aggregations of polymerized actin, a-actinin and tropomyosin above and beside the invading bacterium in both epithelial cell lines. These structures were evident soon after bacterial addition (maximal at 20 min for HeLa cells, 60 min for MDCK cells), and disappeared later in the infection as the cytoskeletal components returned to a more normal distribution after bacterial internalization. Surprizingly, tubulin also aggregated above internalized Salmonella although bacterial entry or penetration through polarized monolayers was not disrupted by the microtubule-inhibiting agent nocadazole (this treatment actually enhanced tubulin accumulation around these organisms). There were little if any rearrangements in intermediate filaments composed of keratin or vimentin. Large amounts of talin also accumulated above and around invading Salmonella, but there was only a minor accumulation of vinculin around a few organisms. Pretreatment of epithelial cells with the microfilament inhibitor cytochalasin D blocked bacterial internalization but did not prevent accumulation of polymerized actin and a-actinin directly beneath uninternalized bacteria, yet prevented accumulation of the other cytoskeletal components. These results suggest that Salmonella bind to the surface and trigger a signal in epithelial cells that causes marked rearrangements in various cytoskeletal components, including recruitment of actin filaments and a-actinin, which then generates the force necessary for bacterial uptake.
Chemoreceptor-mediated elongation of stereocilium bundles tunes vibration-sensitive mechanoreceptors on cnidocyte-supporting cell complexes to lower frequencies
Authors: Watson Glen M. , Hessinger David A.
Published: April 2021

Cnidocyte-supporting cell complexes (CSCCs) discharge nematocysts into targets upon coincidental stimulation of specific chemoreceptors and contactsensitive mechanoreceptors. In addition, CSCCs in the tentacles of at least one species of sea anemone discharge nematocysts into targets vibrating at specific frequencies. In seawater alone, these CSCCs discharge nematocysts preferentially at 55, 50 and 75 Hz. In the presence of 10−7M N-acetylneuraminic acid (NANA) or mucin, the CSCCs discharge nematocysts preferentially at the lower frequencies of 0, 5, 15, 30 and 40 Hz. Furthermore, the stereocilium bundles (SBs) within ciliary cones of CSCCs elongate significantly from a mean length of 6.08 /on in seawater to 7.14 μm in 10−7M mucin. The responses of (1) shifting the optimal frequencies for discharging nematocysts to lower frequencies and (2) elongating the SBs both exhibit dose-dependency and temporal adaptation to chemosensitizer. We conclude that these responses are controlled by CSCC chemoreceptors for N-acetylated sugars. We suggest that specific size-classes of SBs respond to specific frequencies of vibration, since the dose-response parameters to NANA depicting the relative abundances of SB size classes measuring 3–4, 5 and 7 μm correlate with dose-response parameters for the discharge of nematocysts into targets vibrating at 75, 55, and 30 Hz. Treating tentacles with cytochalasin disorganizes the SBs of ciliary cones and decreases the number of frequency optima for nematocyst discharge without significantly affecting nematocyst discharge into static targets. Thus, ciliary cones on CSCCs are vibration-sensitive mechanoreceptors that can be tuned by chemoreceptors to specific, lower frequencies by the elongation of SBs.
Effects of truncated neurofilament proteins on the endogenous intermediate filaments in transfected fibroblasts
Authors: Chin Steven S. M. , Macioce Pompeo , Liem Ronald K. H.
Published: April 2021

The expression and assembly characteristics of carboxyl- and amino-terminal deletion mutants of rat neurofilament low Mr (NF-L) and neurofilament middle Mr (NF-M) proteins were examined by transient transfection of cultured fibroblasts. Deletion of the carboxyl-terminal tail domain of either protein indicated that this region was not absolutely essential for co-assembly into the endogenous vimentin cytoskeleton. However, deletion into the α-helical rod domain resulted in an inability of the mutant proteins to co-assemble with vimentin into filamentous structures. Instead, the mutant proteins appeared to be assembled into unusual tubular-vesicular structures. Additionally, these latter deletions appeared to act as dominant negative mutants which induced the collapse of the endogenous vimentin cytoskeleton as well as the constitutively expressed NF-H and NF-M cytoskeletons in stably transfected cell lines. Thus, an intact a-helical rod domain was essential for normal IF co-assembly whereas carboxyl-terminal deletions into this region resulted in dramatic alterations of the existing type HI and IV intermediate filament cytoskeletons in vivo. Deletions from the amino-terminal end into the a-helical rod region gave different results. With these deletions, the transfected protein was not co-assembled into filaments and the endogenous vimentin IF network was not disrupted, indicating that these deletion mutants are recessive. The dominant negative mutants may provide a novel approach to studying intermediate filament function within living cells.
Expression in escherichia coli of human lamins A and C: influence of head and tail domains on assembly properties and paracrystal formation
Authors: Moir Robert D. , Donaldson Anne D. , Stewart Murray
Published: April 2021

We have expressed in Escherichia coli cDNA corresponding to human lamins A and C, together with a number of fragments produced using site-specific mutagenesis. The proteins produced in this way were characterised both biochemically and ultrastructurally, and appeared to retain their native conformation. Crosslinking showed that all fragments formed 4-chain molecular dimers (‘tetramers’) analogous to those formed by intact intermediate filament proteins. Shadowed preparations showed the presence of rod-like particles that closely resembled those observed for other intermediate filament proteins and their proteolytically prepared rod domains. Moreover, the expressed lamins and a series of fragments in which different domains had been deleted formed paracrystals similar to those observed with native material. Deletion of either the Nor C-terminal non-helical domains altered the solubility and aggregation properties of the expressed protein, indicating that both domains have a role in lamin assembly.