bluuebunny/crossref_metadata_2025_split huggingface dataset:
Studies of chemotactic factor-induced polarity in human neutrophils : Lipid mobility, receptor distribution and the time-sequence of polarization
Authors: McKAY D. A. , Kusel J. R. , Wilkinson P. C. 
Published: April 2021

ABSTRACT
Differences in membrane composition between the anterior and posterior poles of human blood neutrophils on exposure to chemoattractant were studied using a laser microscope, and the effects of attractant concentration and time on the ability of neutrophils to polarize were determined. The findings were as follows. (1) The fluorescein-labelled chemotactic hexapeptide fLeu-Norleu-Phe-Norleu-Tyr-Lys was asymmetrically distributed on polarized cells with the highest concentration at the anterior pole of the cell. (2) Differences in membrane lipid behaviour were studied by fluorescence recovery after photobleaching (FRAP) using the probe 5-N-(octadecanoyi)-aminofluorescein. Recovery curves suggested that the proportion of mobile lipid probe at the anterior pole of the cell was higher than at the posterior. However, no difference was found in the rate of recovery between the two poles. (3) Studies of the time-course of polarization were undertaken using neutrophils in suspension exposed to different isotropic concentrations of fMet-Leu-Phe (fMLP). At low concentrations (≤10-9M), the cells polarized immediately on exposure to the attractant. At a high concentration (10-7M) they assumed multipolar morphologies and polarized very poorly, suggesting that ligand binding was too rapid for the cells to form a dominant pole. At the optimal concentration, 10-8M fMLP, the cells assumed irregular, ruffled, morphologies after 30 s, but showed an increasing proportion of polarized forms over the next 30 min. These results suggest that, while cells exposed to low concentrations of attractant may respond by polarizing towards the site of a first signal from ligand, at optimal attractant concentrations a dominant pole is formed by integration of responses to ligand-binding at multiple sites. The polarization response is likely to be a stochastic process determined by chance events such as asymmetry or fluctuations in ligandreceptor binding.
Epidermal growth factor induces rapid reorganization of the actin microfilament system in human A431 cells
Authors: Rijken Philip J. , Hage Willem J. , Henegouwen Paul M. P. Van Bergen En , Verkleiji Arie J. , Boonstra Johannes 
Published: April 2021

ABSTRACT
Double immunofluorescence microscopy reveals that epidermal growth factor (EGF) treatment of A43l cells results in more apparent co-localization of EGF receptor (EGFR) and actin fllaments, as compared to control cells. This indicates that EGF induces actin polymerization as well as additional association of the EGFR with similar sites on the membraneskeleton. We show that immunoprecipitation of the cytoskeleton-linked EGFR after fragmentation of the cytoskeleton results in specific co-precipitation of F-actin and a limited set of other unidentified proteins. Interestingly, EGF treatment of intact cells results in increased immunoprecipitation of cytoskeleton-associated EGFR as well as of F-actin, while actin does not co-precipitate with the non-ionic detergent-solubilized EGFR. These results demonstrate that the cytoskeleton-linked EGFR is associated with the actin microfilament system. EGF induces additional formation of protein complexes, containing the EGFR and F-actin and a limited set of other unidentified proteins. The increased co-pre-cipitation of F-actin is most likely related to EGF-induced actin polymerization, which is specifically associated with the apical cortical microfilament system, as demonstrated by confocal laser scanning microscopy and a phallicidin-binding assay.
Physarum plasmodia do contain cytoplasmic microtubules!
Authors: Salles-Passador Isabelle , Moisand André , Planques Viviane , Wright Michel 
Published: April 2021

ABSTRACT
It has been claimed that the plasmodium of the myxomycete Physarum polycephaium constitutes a very unusual syncytium, devoid of cytoplasmic microtubules. In contrast, we have observed a cytoplasmic microtubule network, by both electron microscopy and immunofluorescence in standard synchronous plasmodia, either in semi-thin sections or in smears, and in thin plasmodia, used as a convenient model. Cytoplasmic microtubules could be seen after immunofluorescent staining with three different monospecific monoclonal anti-tubulin antibodies. The immunolabelling was strictly restricted to typical microtubules as shown by electron microscopy. These cytoplasmic microtubules were entirely and reversibly disassembled by cold treatment and by either of two microtubule poisons: methyl benzimidazole carbamate and griseofulvin. The microtubule network, present in all strains that have been studied, contains single microtubules and microtubule bundles composed of two to eight microtubules. Cytoplasmic microtubules form a dense and complex three-dimensional network, distinct from the microfilamentous domains and from the nuclei. The orientation of the microtubule network varies according to the plasmodial domain examined. Generally microtubules show no special orientation except in plasmodial veins where they are oriented parallel to the long axis of the veins. Differences between our observations and those of previous workers who failed to find cytoplasmic microtubules in plasmodia are discussed. We propose that they reflect difficulties of observation mainly due to the fluorescent background. In contrast with the previous view, the discovery of a microtubule cytoplasmic cytoskeleton in Physarum plasmodia raises several questions concerning its relationships with other cellular organelles and its dynamics during different cell cycle events.
Tubulin in sea urchin embryonic cilia: characterization of the membrane-periaxonemal matrix
Authors: Stephens R. E. 
Published: April 2021

ABSTRACT
When the membranes of isolated embryonic cilia from three species of sea urchin are detergent-solubilized, the major proteins found are two equimolar polypeptides comigrating with tubulin subunits. Cross-reactivity with a variety of tubulin antibodies confirms their identity. Calmodulin and other calcium-binding proteins are prominent minor constituents of the extract. Removal of the solubilizing detergent by adsorption to polystyrene beads, followed by a freeze-thaw cycle, produces membrane leaflets and vesicles of uniform bouyant density. Such reconstituted membranes incorporate most of the tubulin and minor proteins but not calmodulin. Equivalent cross-reactivity with antibodies to acetylated or detyrosinated α-tubulin indicates that the tubulin derived from the mem-brane-periaxonemal matrix and axoneme are indistinguishable from each other in terms of these post-translational modifications but are distinct from the mainly unmodified tubulin of the embryonic cytoplasm. Pulse labeling with [3H]palmitate does not label either tubulin subunit but acylation does occur on a 190 ×103Mr membrane protein. Its specific activity is essentially the same whether label is applied to embryos with existing or regenerating cilia, suggesting rapid ciliary membrane protein exchange or physical turnover. Using pulse-chase labeling with [3H]leuc?ne during steady-state ciliary growth or induced regeneration in both normal and zinc-animalized embryos, the specific activity of the membrane-periaxonemal matrix-derived tubulin is initially higher than that of the axoneme but the degree of labeling equalizes in successive regenerations, consistent with derivation from a common pool. Many heavily labeled axonemal architectural proteins, such as tektin-A, are not reflected in the membrane-periaxonemal matrix fraction, suggesting that this fraction is not simply a pool of unassembled axonemal precursors.
Differential pathways of recruitment for centrosomal antigens to the mitotic poles during bipolar spindle formation
Authors: Maekawa Takami , Kuriyama Ryoko 
Published: April 2021

ABSTRACT
As cells enter mitosis, centrosomes undergo many transformations and become associated with different molecules in a stage-specific manner. We have developed a protocol for immunofluorescence staining with four antibody probes that can help us to follow the interaction of centrosomal components during mitosis. The cells were first stained with a human autoimmune serum (5051); a monoclonal anti-phosphocentrosomal antibody (CHO3); and an antitubulin antibody. Localization of the antibodies was detected using rhodamine-, fluorescein- and AMCA-conjugated second antibodies, respectively. After photographing marked mitotic cells, coverslips were soaked with 0.2 M glycine–HCl at pH 1.0 for lh to release all antibodies bound to the structures. The same cells were re-stained with a human autoantibody (SP-H) specific for spindle poles and a fluor-escein-conjugated second antibody. This allowed us to compare the subcellular distribution of three kinds of centrosomal antigens in a single cell. Mitotic PtKi cells treated with either nocodazole or taxol included microtubule-containing cytoplasmic foci and parallel bundles of short microtubules at the cell periphery. All the centrosomal antibodies stained the same one or two dots corresponding to structures labeled by the tubulin antibody. CHO3 also revealed extra cytoplasmic foci, whereas the SP-H antigen was additionally localized at one end of the free microtubule bundles. As the microtubules reorganized into bipolar spindles during the recovery from drug treatment, the CHO3 and SP-H antigens coalesced into the spindle poles where the 5051 antigen was located, suggesting that centrosomal antigens become associated with spindle poles through very different recruitment pathways.
Experimental obliteration of the preprophase band alters the site of cell division, cell plate orientation and phragmoplast expansion in Adiantum protonemata
Authors: Mineyuki Yoshinobu , Murata Takashi , Wada Masamitsu 
Published: April 2021

ABSTRACT
Division sites in higher plant cells are known to be determined before mitosis, and cell plates are precisely inserted into the predetermined division sites at the end of cytokinesis by unknown mechanisms. When apically growing protonemal cells of the fern, Adiantum capillus-veneris L., grown under red light are transferred to the dark, apical growth ceases and the protonemal cells then divide. However, this mitotic event can be influenced by subsequent exposure to light. If red-light pre-cultured protonemata are incubated in the dark and then transferred back to red light (after 28–36 h), apical growth resumes and the nuclei migrate toward the growing tips; interestingly, mitosis still occurs, although in an altered fashion. In the re-irradiated cells, timing of the premitotic nuclear positioning is delayed markedly and irregularly oriented cell plates are frequently observed. Re-irradiation with red light also causes an increase in cells without preprophase bands (PPBs) at prophase and the irregular expansion of the phragmoplast at late telophase, while early phragmoplast microtubule (MT) organization takes place normally. These data suggest the indirect involvement of PPBs in the guidance of phragmoplast expansion.
Topography of connexin32 in rat liver gap junctions : Evidence for an intramolecular disulphide linkage connecting the two extracellular peptide loops
Authors: Rahman Salman , Evans W. Howard 
Published: April 2021

ABSTRACT
A range of anti-peptide antibodies directed towards selected amino acid sequences of connexin32 was prepared and characterised. The site-directed antibodies that identified connexin32 were used to study by immunolocalization and by proteolytic treatment of intact and split gap junctions the arrangement of the protein in the membrane. These studies reinforce models of connexin topography in which the polypeptide traverses the junctional membrane four times, with the amino and carboxyl termini cytoplas-mically located. The four transmembrane domains were shown to be linked by two extracellular loops with a single intracellular loop connecting the second and third transmembrane domains. Evidence is presented to show that the two extracellular domains of connexin32, which are important for intercellular adhesion and the insulated bridging of the extracellular space by channels allowing cellcell communication across the gap junction, are connected by disulphide bond(s). The studies lead to a more detailed two-dimensional model of con-nexin32 in the membrane, incorporating the favoured theoretical arrangement of disulphide bonds at the extracellular domain of connexin32.
Distribution of fibronectin and fibronectin-binding proteins, AGp110 and integrin α
5
β
1 during chemically induced hepatocarcinogenesis in adult rats
Authors: Stamatoglou Stamatis C. , Manson Margaret M. , Green Jonathan A. , Mayol Xavier , Hughes R. Colin 
Published: April 2021

ABSTRACT
We have used single- and double-label immunocytochemistry to examine the distribution of AGpllO, integrin α5β1 and fibronectin in adult rat liver during carcinogenesis induced by aflatoxin B2 or diethyl-nitrosamine. In normal liver fibronectin and the fibronectin integrin receptor α5β1 are localized on all three domains of the parenchymal cell surface: sinusoidal, lateral and canalicular. In contrast, AGpllO, a non-integrin monomeric glycoprotein with fibronectin receptor properties, is confined to the bile canalicular (apical) plasma membrane of hepatocytes. Hepatocarcinogenesis induced by aflatoxin B2 causes altered cell foci to form in the parenchyma, followed by enlargement of these foci to form pre-neoplastic nodules and finally hepatocellular carcinomas of either poorly differentiated, trabecular or adenocarcinoma morphology. Expression of AGpllO decreased to a minimal level, at first selectively in altered cell foci, from the 9th week of treatment, and then indiscriminately in poorly differentiated carcinomas. The same lesions that were deficient in AGpllO also displayed a reduced level of fibronectin and 25βi, although the observed change in AGpllO demarcated altered foci and poorly differentiated tumour lesions more sharply, since expression of α5β1 and fibronectin, though substantially reduced, was still faintly apparent on the cell surface. Small acinar structures, observed in late hyperplastic nodules and in trabecular carcino mas, exhibited even, pericellular staining of fibronectin and α5β1 including prominent staining of the lumen area, whereas staining of AGpllO appeared to be confined to the lumen. In larger ducts of overt adenocarcinomas, fibronectin and α5β1 were distributed along the basal surface of the epithelium and AGpllO on the apical domain. Tumours induced by diethylnitrosamine and promoted with ethinyl estradiol displayed similar histology and staining patterns for all three proteins as that described for aflatoxin B2. Finally, comparisons between AGpllO and cytokeratin 19, a selective tumour marker, indicated that whereas loss of AGpllO occurs in poorly differentiated lesions and tumours, expression of cytokeratin 19 is associated with acinar and glandular structures found in late hyperplasia and with trabecular and pseudoglandular tumours. The results indicate that loss of differentiation in either hyperplastic or neoplastic lesions correlates with reduced expression of fibronectin and of its receptors α5β1 and AGpllO. On the basis of morphological similarities and staining patterns in the pre-neoplastic and neoplastic state, we deduce that hepatocellular carcinomas derive from differentiated hepatocytes.
Characterisation of potential adhesins of the bacterium Pasteuria penetrans, and of putative receptors on the cuticle of Meloidogyne incognita, a nematode host
Authors: Lay A. Persidis, J. G. , Manousis T. , Bishop A. H. , Ellar D. J. 
Published: April 2021

ABSTRACT
Pasteuria penetrans spores were fragmented by glass bead vortexing, producing exosporiai membranes and spore fragments, which consisted of fibre bundles. Both exosporia and spore fragments are capable of host-specific attachment to the cuticle of Meloidogyne incognita, a root-knot nematode host. Putative M. incognita receptors appear to be soluble in β-mercaptoethanol (BME) but not SDS, and are also sensitive to tryptic digestion and deglycosylation by endoglycosidase F. Polyclonal antibodies against intact spores and spore fragments inhibited attachment up to 25% and Fab’ fragments of antispore antibodies produced 100% inhibition. The antibodies, however, did not show preferential staining of particular spore structures in thin section immunolabelling studies. Exposure of Pasteuria penetrans spores to HC1 or urea-SDS-dithiothreitol renders them incapable of attachment to their host juveniles and extensively disrupts fibres that surround the spore core. Protein extracts from spore fragments or from exosporiai membranes are identical, and urea-BME extracts from either structure, but not SDS extracts, can inhibit the attachment of spores to juveniles by 60-80%. An inhibitory BME extract from spore fragments was analysed by anion-exchange chromatography and adsorption onto host cuticle followed by immunoblotting. It appeared to contain six potential spore adhesins of approximate Mr 24-29, 38-47, 59, 89, 126, and 190 (×IO3). Lectin affinity blotting with wheat germ agglutinin and concanavalin A showed that all of these proteins bear terminal N-acetylglucosamine residues and the 38–47 kDa band also bears terminal Glc/Man residues. A BME extract from strain Pp 1 inhibited the attachment of strain PNG to M. incognita and vice versa and the PNG strain had a greater amount of two proteins of 98 and 126 kDa than the Pp 1 extract.
Hormonal regulation of spermatid binding
Authors: Cameron Don F. , Muffly Karl E. 
Published: April 2021

ABSTRACT
A Sertoli-spermatid coculture model is described in which a large percentage (&gt;76%) of round spermatids remain viable for 48 h and bind to Sertoli cells. The effects of follicle-stimulating hormone (FSH) and testosterone on spermatid binding (expressed as the spermatid density; SD- the number of spermatids per unit area of Sertoli cell cytoplasm), ultrastructure of the Sertoli-spermatid junctional complex, and distribution in the Sertoli cell of junction-related F-actin and vinculin are described. Following 48 h of incubation, neither FSH alone nor testosterone alone affected spermatid binding to Sertoli cells beyond that observed in control cocultures. However, the combination of FSH and testosterone (FSH+testosterone) resulted in a significant increase in the density of spermatids bound to Sertoli cells. Junction-related structure of the Sertoli cell cytoskeleton between the Sertoli cell and the pre-step 8 spermatid was different than that observed between the Sertoli cell and the post-step 8 spermatid. The junction-related cytoskeletal modification of the Sertoli cell (JCMS) in the latter was similar in appearance to the well-described ‘Sertoli ectoplasmic specialization* observed adjacent to post-step 8 spermatids in vivo. FSH+testosterone and FSH alone, but not testosterone alone, resulted in the peripheral distribution of actin and vinculin, which otherwise remained in stress fiber-like structures throughout the Sertoli cell. Results show that maximal spermatid binding to Sertoli cells in vitro requires FSH+testosterone and is associated with the peripheral distribution of actin and vinculin.
Isoproterenol downregulation of statin-related gene expression in the rat parotid gland
Authors: Ann David K. , Wechsler Aharon , Lin H. Helen , Wang Eugenia 
Published: April 2021

ABSTRACT
Statin, a 57 kilodalton (kDa) nuclear protein, is characteristically found in nonproliferating cells in culture as well as nondividing cells of a wide range of highly differentiated tissues. Moreover, cells in culture that are statin positive lose this statin expression when re-entering the cell-cycle traverse. In this work, statin expression was investigated in the parotid gland of untreated rats and those treated with isoproterenol (IPR), a proliferation-inducing catecholamine. Indirect immunofluorescence microscopy revealed specific nuclear staining with antistatin monoclonal antibody (S-44) in the acinar and ducts cells of the untreated rats but significantly reduced in those induced with isoproterenol. To characterize the protein recognized by S-44, protein extracts from both tissues were immunoblotted and incubated with S-44. The antibody reacted specifically with a 48 kDa protein in the extract of the parotid glands from untreated rats while no reaction was detected in that of the proliferation-induced ones. These observations along with the result that a statin-like (SI) transcript is downregulated by isoproterenol in the parotid glands further support the notion that the disappearance of statin-related expression is associated with the IPR-induced proliferation in the rat parotid glands. The discrepancy between the apparent molecular mass of the protein identified by S-44 in nonproliferating parotid cells and that of statin originally found in fibroblasts, suggests that either a modified form of statin may be present in the parotid gland, or this 48 kDa protein may be a member of the nonproliferative statin-like family.
Dominance of a NOR (nucleolar organizer region) over its allele and over its sister NOR after asymmetric 5-azacytidine substitution in plant chromosomes
Authors: Torre C. De La , Gimenez-Abian J. F. , Gonzalez-Fernandez A. 
Published: April 2021

ABSTRACT
Dominance of a 5-azacytidine-substituted nucleolar organizer region (AZA NOR) over another with normal DNA when confined to a common nucleus or to different nuclei in a common cell was studied in Allium cepa L. root meristems. AZA administration took place for one S period. The assessment of NOR dominance was done either in early Gi (nucleologen-esis) or later in interphase (mature nucleoli). At the mitosis immediately preceding the second interphase after AZA, the segregated AZA and normal chromatids of each chromosome of the pair of homologues were confined to a single binucleate cell by inhibiting the formation of the cell plate. Anaphase segregation of the homologous AZA NORs was seen to occur at random.
The AZA NORs completed nucleologenesis after a shorter time than normal NORs, independently of the intranuclear or intracellular presence of other NORs. After nucleologenesis was over in the AZA NORs, a few of the normal NORs, when in a common nucleus, remained permanently inefficient in developing a full-size mature nucleolus or any nucleolus at all (partial or total dominance, respectively).
Dominance of an AZA NOR over its sister or allelic NOR with normal DNA led to the new nucleolar patterns of asymmetry and inverse symmetry, respectively, making their appearance in these binucleate cells.
How relevant is the Escherichia coli uvrabc model for excision repair in eukaryotes?
Authors: Hoeijmakers J. H. J. 
Published: April 2021

ABSTRACT
Knowledge about the DNA excision repair system is increasing rapidly. A detailed model for this process in Escherichia coli has emerged in which a lesion in the DNA is first recognized by the UvrA2B helicase complex. Subsequently, UvrC mediates incision on both sites of the DNA injury. Finally, the concerted action of helicase II (UvrD), polymerase and ligase takes care of removal of the damage-containing oligonucleotide, DNA resynthesis and sealing of the residual nick.
In the eukaryotes, yeast and mammals a total of 10 excision repair genes have been analysed thus far. However, little is still known about the molecular mechanism of this repair reaction. Amino acid sequence comparison suggests that at least three DNA helicases operate in eukaryotic nucleotide excision. In addition, a striking sequence conservation is noted between human and yeast repair proteins. But no eukaryotic homologs of the UvrABC proteins have been identified. In this Commentary the parallels and differences between the prokaryotic and eukaryotic excision repair pathways are weighed in an attempt to assess the relevance of the E. coli model for the eukaryotic system.