bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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.
A comprehensive study on the isolation and characterization of the hela S3 nuclear matrix
Authors: Belgrader Phillip , Siegel Alan J. , Berezney Ronald 
Published: April 2021

ABSTRACT
Different agents have been employed to extract the histones and other soluble components from isolated HeLa S3 nuclei during nuclear matrix isolation. We report that 0.2 M (NH4)2SCL is a milder extracting agent than NaCl and LIS (lithium 3,5-diiodosali-cylate), on the basis of the apparent preservation of the elaborate fibrogranular network and the residual nucleolus that resemble the in situ structures in whole cells and nuclei, minimal aggregation, and sufficient solubilization of DNA and histones.
The importance of intermolecular disulfide bonds, RNA and 37 °C stabilization on the structural integrity of the nuclear matrix was examined in detail using sulfydryl alkylating, reducing and oxidizing agents, and RNase A. The data suggest that any disulfides formed during the isolation are not essential for maintaining the structural integrity of the in vitro matrix. However, structural integrity of the matrix is dependent upon RNA and to some degree on disulfides that presumably existed in situ. Sodiumtetrathionate and 37 °C stabilization of isolated nuclei resulted in nuclear matrices containing an approximately twofold greater amount of protein, RNA and DNA than control preparations. The 37°C incubation, unlike the sodium tetrathionate stabilization, does not appear to induce intermolecular disulfide bond formation. Neither stabilizations resulted in significant differences of the major matrix polypeptide pattern on two-dimensional (2-D) gels stained with Coomassie Blue as compared to that of unstabilized matrix.
The major nuclear matrix proteins, other than the lamins, did not react to the Pruss murine monoclonal antibody (IFA) that recognizes all known intermediate filament proteins, suggesting that the internal matrix proteins are not related to the lamins in intermediate filament-like quality.
Monoclonal antibodies to plant nuclear matrix reveal intermediate filament- related components within the nucleus
Authors: Beven Alison , Guan Yuhong , Peart Jan , Cooper Christine , Shaw Peter 
Published: April 2021

ABSTRACT
We have prepared a nuclear matrix fraction from purified nuclei of carrot (Daucus carota L.) suspension culture cells, and used this fraction to produce a library of monoclonal antibodies. We report the preliminary characterisation of two antibodies – JIM 62 and JIM 63. The antibodies recognise a polypeptide doublet band at 92×103Mr, which has been partially purified by differential urea extraction. Other intermediate filament antibodies – ME 101, which recognises an epitope conserved among many intermediate filament proteins, and AFB, a monoclonal antibody to plant intermediate filament proteins, and an autoimmune serum directed against human lamins A and C (LSI), also label these bands, suggesting they are related to the intermediate filament/lamin family. IFA, another intermediate filament antibody, labels a band at approximately 60×103Mr which is also enriched in the urea extracts of nuclear matrices. Immunofluorescence microscopy with JIM 63, ME 101, AFB and LSI shows network-like staining, often extending around the nucleolus. In many cases the staining reveals structures that appear to be bundles of fibres. JIM 63 also shows a weak staining of the nuclear rim in carrot nuclei, which can be greatly enhanced by treatment of the specimen with cold methanol after fixation. JIM 63 cross-reacts with all the other plant species we have tested. Vibratome sections of pea roots, extracted as for nuclear matrix preparation and stained with JIM 63 show a clear, strong nuclear rim labelling. Furthermore, JIM 63 strongly labels the nuclear lamina in rat liver nuclei. We suggest that the 92×103Mr protein(s) are related to intermediate filaments and/or lamins, and are distributed both within the nucleus and at the nuclear periphery.
Evidence that intermediate filament reorganization is induced by ATP-dependent contraction of the actomyosin cortex in permeabilized fibroblasts
Authors: Tint I. S. , Hollenbeck P. J. , Verkhovsky A. B. , Surgucheva I. G. , Bershadsky A. D. 
Published: April 2021

ABSTRACT
Intermediate filaments (IFs) undergo specific rearrangements in cells, some aspects of which can be induced experimentally. Centripetal aggregation of the IF network, for example, can be produced by a variety of perturbations. However, the source of motive force is clear for neither in vivo nor experimentally generated IF movements, since, unlike microtubules and actin filaments, IFs have no known force-generating system directly associated with them. We recently obtained evidence that the drug-induced aggregation of vimentin IFs in fibroblasts is an active event, which requires ATP and involves the actin cytoskeleton. In the present study, we sought to test the hypothesis that IF aggregation is driven by a centripetally directed contraction of the actomyosin cortex. To that end, we have permeabilized fibroblasts with Triton X-100 in a stabilizing buffer and reactivated cytoskeletal movements in vitro, under defined solution conditions. Upon nucleotide treatment, these permeabilized cells undergo a nucleotide-dependent centripetal aggregation of vimentin IFs similar in appearance and time course to that induced in intact cells by drug treatment. During in vitro IF aggregation, the permeabilized cells remain fully spread and adherent to the substratum, and the distal ends of the microtubules and actin microfilaments retain their positions in the cell periphery. IF aggregation is accompanied by a contraction of F-actin and myosin into focal aggregates in the same perinuclear region in which the IFs accumulate. If permeabilized cells are treated with the actin-severing protein gelsolin prior to the reactivation of IF movement, the actin cytoskeleton is eliminated and IF aggregation fails to occur when ATP is added. These results strongly support a model in which the motive force for IF movement is supplied indirectly by association with a contracting actomyosin network.
Purification and physical properties of nematode mini-titins and their relation to twitchin
Authors: Nave Rüdiger , Fürst Dieter , Vinkemeier Uwe , Weber Klaus 
Published: April 2021

ABSTRACT
We have isolated mini-t Ítin from the nematodes Ascaris hunbricoides and Caenorhabditis elegans under native conditions using a modification in the procedure to prepare this protein from insect muscle. The proteins have an apparent molecular weight of 600 000 and appear in oriented specimens as flexible thin rods with a length around 240–250 nm. The circular dichroism spectrum of the Ascaris protein is dominated by β-structure. The proteins react with antibodies to insect mini-titin and also with antibodies raised against peptides contained in the sequence predicted for twitchin, the product of the Caenorhabditis elegans unc-22 gene. Antibodies to insect mini-titin decorate the body musculature as well as the pharynx of wild-type C. elegans in immunofluorescence microscopy. In the twitchin mutant E66 only the pharynx is decorated. We conclude that the mini-titins of invertebrate muscles defined earlier by ultrastructural criteria are very likely to be twitchins, i.e. molecules necessary for normal muscle contraction. We discuss the molecular properties of the proteins in the light of the sequence established for twitchin.