bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conjugation of Paramecium tetraurelia cells: selective wheat germ agglutinin binding, reversible local trichocyst detachment and secretory function repair*
Authors: Pape Regina , Haacke-Bell Birgit , Lüthe Norbert , Plattner Helmut 
Published: April 2021

ABSTRACT
Conjugation of ciliated protozoa involves precisely defined stages. In Paramecium there occur: (1) the formation of ciliary contacts; (2) local loss of cilia and formation of narrow contacts between anterioventral somatic cell membranes; followed by (3) formation of small cytoplasmic bridges (which allow for the exchange of molecular components only); before (4) their enlargement enables the cells to exchange micronuclei. This work concentrates on stages (2) and (3) in P. tetraurelia cells. We analysed lectin binding during conjugation, using mainly secretory mutants nd6 and tl (occasionally nd9-28°C and 7S (wildtype) cells); (tl is a trichocyst-free mutant, nd strains are non-discharge mutations). Our most essential findings are: already early contact sites (stage (2)) can be labelled with wheat germ agglutinin-fluorescein isothiocyanate; only early stages (2) can be reversed with an excess of WGA; no selective labelling was found with concanavalin A-fluorescein isothiocyanate (commonly observed with other ciliate species); WGA binding sites most probably involve GlcNAc residues. As known before, trichocysts disappear from contact sites in stages (2)/(3). We then analysed this process in more detail. Trichocysts are locally detached from the cell membrane (without transfer to the other conjugant) and re-inserted after conjugation. From our data obtained with nd6μl con jugants one can conclude that trichocysts (in particular secretory lectins or glycoproteins contained in them) cannot play a regulatory role in the conjugation process, since conjugation precedes secretory function repair. We also present evidence for a secretory function repair in nd mutations by tl cells (though tl cells are devoid of any recognizable ‘exocytotic apparatus’). nd6 cells are more difficult to ‘cure’ than nd9-28°C. The ‘curing effect’ seems to emanate from the conjugation zone. This might indicate the occurrence of diffusible ‘curing factors’ exchanged mainly in stage (3). Finally, we show that secretory function repair in nd cells induces the assembly of ‘fusion rosettes’, i.e. the formation of the wild-type ultrastructure of the trichocyst release sites, to about the same extent as nd cells are functionally repaired (visualized by the release of trichocysts in response to two different trigger agents).
Ubiquitin gene expression in Dictyostelium is induced by heat and cold shock, cadmium, and inhibitors of protein synthesis
Authors: Müller-Taubenberger A. , Hagmann J. , Noegel A. , Gerisch G. 
Published: April 2021

ABSTRACT
Ubiquitin is a highly conserved, multifunctional protein, which is implicated in the heat-shock response of eukaryotes. The differential expression of the multiple ubiquitin genes in Dictyostelium discoideum was investigated under various stress conditions. Growing D. discoideum cells express four major ubiquitin transcripts of sizes varying from 0·6 to 1·9 kb. Upon heat shock three additional ubiquitin mRNAs of 0·9, 1·2 and 1·4 kb accumulate within 30 min. The same three transcripts are expressed in response to cold shock or cadmium treatment. Inhibition of protein synthesis by cycloheximide leads to a particularly strong accumulation of the larger ubiquitin transcripts, which code for polyubiquitins. Possible mechanisms regulating the expression of ubiquitin transcripts upon heat shock and other stresses are discussed.
Formation of plasma membrane domains in rat hepatocytes and hepatoma cell lines in culture
Authors: Maurice M. , Rogier E. , Cassio D. , Feldmann G. 
Published: April 2021

ABSTRACT
In vivo, proteins of the hepatocyte plasma membrane are asymmetrically distributed, making it possible to distinguish a sinusoidal, a lateral and a canalicular domain. The conditions that determine hepatocyte plasma membrane polarity have been investigated in vitro, using three monoclonal antibodies directed against integral membrane proteins, which were characteristic of each domain. The localization of the three antigens was studied by immunolabelling of hepatocytes isolated from adult rat liver, primary monolayer cultures and rat hepatoma cell lines. When hepatocytes were isolated, the three antigens spread over the entire cell surface. The lateral antigen redistributed at lateral sites as soon as cell-cell contacts were established, 4h after the beginning of primary culture. The sinusoidal and canalicular antigens became asymmetrically distributed after 48 h of primary culture, after the formation of bile canaliculus-like structures. In most of the hepatoma lines studied, the three antigens were expressed, except that the canalicular antigen was fully expressed in differentiated clones only. The lateral antigen was always distributed on thecontiguous membranes of clustered hepatoma cells, whereas the sinusoidal and canalicular antigens were localized on the entire plasma membrane. However, in a few cells of some clones in which bile canaliculus-like structures were observed, the canalicular membranes were strongly labelled only with the canalicular antibody. In the absence of bile canalicular formations, in both primary culture and cell lines, the canalicular antigen and, to a lesser extent, the sinusoidal antigen accumulated in the Golgi apparatus, suggesting that their transport to the cell surface was altered in the absence of a bile pole. These results show that in hepatic cells, polarization of the plasma membrane is determined by: (1) the existence of cell-cell contacts, which is correlated with the domain-specific localization of the lateral antigen; and (2) the formation of bile canaliculi, which would trigger the development of an asymmetrical distribution of the sinusoidal and canalicular antigens.
Effect of enzymic (collagenase) harvesting on the intracellular Na+/k+ Ratio of swiss/3t3 cells as revealed by x-ray microanalysis
Authors: Szállási Z. , Szállási Á. , Boján F. , Zs.-Nagy I. 
Published: April 2021

ABSTRACT
Swiss/3T3 cell cultures were harvested with 0·05% collagenase and after centrifugation the pellet was prepared by the freeze-fracture/freeze-drying (FFFD) method for bulk-specimen X-ray microanalysis. Time-dependent variations in the intracellular monovalent elemental concentrations (Na+, K+ and Cl-) as well as of the Na+/K+ ratio were followed for 120 min subsequent to harvesting. The quantitative measurements revealed a very considerable increase in the intracellular Na+ and Cl- accompanied by a decrease in the K+ concentration as soon as 5 min after harvesting. The Na+/K+ ratio had increased by this time to about 1-5 on average. These changes indicate a sustained depolarization of the cell membrane. During the first 60 min this depolarization tended to normalize as demonstrated by an exponential decrease in the intracellular Na+ and Cl- and an increase in the K+ content involving a decrease in the Na+/K+ ratio. The total intracellular monovalent ion concentration remained almost constant during this post-harvesting period. These results suggest that harvesting represents a serious depolarizing stimulus to the cells, the consequences of which are restored only after 1·2 h. These alterations should be taken into consideration during various experimental designs when using anchorage-dependent cell cultures.
The role of transport signals and retention signals in constitutive export from animal cells
Authors: Cutler D. F. 
Published: April 2021

ABSTRACT
The question: ‘Do proteins using the constitutive secretory pathway need signals to be retained or signals to be transported?’, has been a fertile source of speculation over the last decade or so. This is at least partly because little direct evidence bearing on the problem has been available until recently. The speculators have fallen into two groups, whose positions can be oversimplified as follows: (1) The secretory pathway is like a conveyer-belt to which a protein gains access through possession of a signal that couples translation to translocation into the endoplasmic reticulum (the signal sequence). Once in the ER, the protein will be carried passively to the cell surface. Membrane proteins will end up in the plasma membrane, soluble proteins will be secreted. To have an intracellular final destination, a signal of some kind that retains the protein within the cell must be present on the transported protein (a ‘retention signal’). (2) The secretory pathway is made up of a number of discrete steps. To move from one organelle to the next along this pathway, a positive signal of some kind must be present on the protein. This signal interacts with a ‘receptor’ of some kind, which mediates its transport at each step (a ‘transport signal’).
Evidence against a direct role for cortical actin arrays in saltatory organelle motility in hyphae of the fungus Saprolegnia ferax
Authors: Heath I. Brent 
Published: April 2021

ABSTRACT
Actin stained by rhodamine-labelled phalloidin in growing hyphae of the oomycete fungus Saprolegnia ferax is restricted to an approximately 0–25 μm deep layer of the cell periphery where it forms an apical cap of fine filaments and a subapical array of coarser longitudinal fibrils interspersed with plaques. The functions of this actin are unknown, but because actin-rich fibrils in other cells are involved in organelle motility I have sought evidence for a similar role in these hyphae. The most prominent motile structures are a population of spherical, predominantly sub-micrometre diameter, refractile vesicles of unknown function, which show typical saltatory movements along the hyphae. The motility of these vesicles is statistically identical in both the central cytoplasm, remotefrom the actin fibrils, and the peripheral cytoplasm adjacent to the fibrils. Treatment of hyphae with specific concentrations and durations of the detergents Tween 20 and Brij 58 causes extensive reorganization of the actin arrays with no effect on vesicle motility, whereas Triton X-100 severely inhibits motility with little detectable effect on the actin filaments. These observations show that normal vesicle motility does not depend on the proximity of a normal population of actin filaments and, therefore, suggest that the latter have some function other than a direct role in saltatory organelle motility.
Electron spectroscopic imaging of the centrosome in cells of the indian muntjac
Authors: Rattner J. B. , Bazett-Jones D. P. 
Published: April 2021

ABSTRACT
Specific antibody labelling indicates that phosphoproteins are present at microtubule-organizing centres, including the centrosome. We have employed electron spectroscopic imaging techniques that permit high-resolution elemental analysis of thin sections of intact cells to investigate the precise distribution of phosphorus and therefore phosphoproteins at the centrosome of Indian muntjac cells. We report that these proteins are localized to both the pericentriolar matrix and the centriole. The matrix contains an abundance of phosphorus and is associated with microtubule elements.
Within the mature centriole, major structures including the nine triplet blades and linking elements that connect adjacent blades are composed of phosphorylated proteins. In addition, phosphoproteins are abundant at the ends of the centriole, at the interface between the centriole lumen and the pericentriolar environment. From these observations we suggest that phosphoproteins may play both a structural and a functional role within the centrosome region.
Dynamic changes in viscoelastic properties in cytotoxic T-lymphocyte-mediated killing
Authors: Sung Kuo-Li P. , Sung Lanping A. , Crimmins Mary , Burakoff Steven J. , Chien Shu 
Published: April 2021

ABSTRACT
The biophysical properties of cytotoxic T lymphocytes during the killing of their target cells was investigated by using a human cytotoxic T lymphocyte clone, Fl, and the target cell, JY, for which it is specific. In single cytotoxic cell/target cell pairs after their conjugation there are changes in the viscoelastic properties of the target cell in association with the lethal hit delivery and post-binding cytolytic steps. On the basis of these changes in the target cell, the complex cytolytic event can be divided into stages: the viscoelastic coefficients exhibited an initial increase followed by a return to resting values; thereafter these coefficients decreased below control and then rose again prior to lysis. The eventual killing of the target cell involves bubbling and swelling of the nucleus, clustering of granules, damage to the cytoplasmic membrane, cell swelling, and lysis. The viscoelastic changes involved in target cell death suggest the loss of integrity of its cytoskeletal apparatus.
The extracellular matrix of hybrids between melanoma cells and normal fibroblasts
Authors: Copeman Michael C. , Harris Henry 
Published: April 2021

ABSTRACT
It has been shown that when malignant tumour cells are fused with normal fibroblasts the suppression of malignancy in the hybrids is linked to their ability to produce a collagenous extracellular matrix in vivo. When, as a consequence of chromosome loss, segregants arise that reacquire malignancy, these do not produce any detectable matrix. In this paper we examine the main components of the extracellular matrix produced in vitro by hybrids between malignant mouse melanoma cells and normal mouse fibroblasts. Hybrids in which malignancy is suppressed synthesize about ten times as much type 1 procollagen as the malignant segregants derived from them; they also retain more fibronectin in the cell layer and release less protease activity into the medium. Malignant segregants more closely resemble the parental melanoma cells in producing fibronectin and mainly types IV and V procollagen. When hybrid cells in which malignancy is initially suppressed are grown continuously in vitro, the production of type I procollagen declines, and the production of type V procollagen and the release of protease activity into the medium increase. These changes, which are associated with the loss from the hybrid cells of both copies of the chromosome 4 derived from the parental fibroblast, predict the reacquisition of malignancy when the cells are inoculated into mice. It is possible that one gene or set of genes located on chromosome 4 determines both the execution of the fibroblast differentiation programme and the suppression of malignancy.
Non-specific elongation of cell cycle phases by cycloheximide in rat 3Y1 cells, and specific reduction of G1 phase elongation by simian virus 40 large T antigen
Authors: Okuda Atsuyuki , Kimura Genki 
Published: April 2021

ABSTRACT
Partial inhibition of protein synthesis by cycloheximide caused prolongation of Gi, S and G2 phases in rat 3Y1 fibroblasts. In cells expressing simian virus 40 (SV40) large T antigen, by infection with SV40 in the previous generation, the prolongation of G1phase in the presence of cycloheximide was suppressed. However, the prolongation of S and G2 phases in the presence of cycloheximide was not suppressed in cells expressing large T antigen, by infection with SV40 in the current generation. Similarly, when density-arrested cells (cells in G0 phase) were infected with SV40 (either wild-type strain or a mutant deleted in the unique coding region for small t antigen) and reseeded sparsely in the presence of cycloheximide, the cycloheximide-induced delay of entry into S phase was suppressed. In this case, the reduction in [35S]methionine incorporation, that in protein accumulation and that in cell volume increase, were not surmounted by SV40 infection. In T-antigen-negative cells, all the regions in G1 phase seemed to be sensitive to cycloheximide, i.e. they suffered elongation. These results suggest that, in comparison with cells that enter S phase by the action of growth factors, cells expressing large T antigen can enter S phase more efficiently through a quite different process.
Interaction of mouse sperm with purified sperm receptors covalently linked to silica beads
Authors: Vazquez Monica H. , Phillips David M. , Wassarman Paul M. 
Published: April 2021

ABSTRACT
We describe a solid-phase assay that bas permitted further analysis of zona pellucida glycoprotein, ZP3, as sperm receptor and acrosome reaction-inducer during fertilization in mice. The assay employs silica beads that contain epoxy groups to which purified, mouse oocyte ZP3 is covalently linked (ZP3-beads). ZP3-beads were characterized, e.g. by whole-mount autoradiography and flow cytofluorometry, incubated with capacitated mouse sperm under a variety of conditions, and the extent of sperm binding determined by light microscopy. Results of experimenta presented suggest the fol-lowing: (1) sperm bind specifically to ZP3-beads, but not to silica beads either exposed to 2-aminoeth-anol or derivatized with oocyte ZP2, fetuin or bovine serum albumin. (2) In nearly ail cases, only one sperm binds per ZP3-bead and binding occurs via the sperm head. (3) The extent of sperm binding to ZP3-beads is dependent on ZP3 and sperm concentrations, as well as on incubation time and temperature. (4) Sperm binding to ZP3-beads is unaffected by antibodies directed against ZP3, but is inhibited in a reversible manner by treatment of ZP3-beads with galactose oxidase. (5) Only acro-some-intact sperm bind to ZP3-beads but, once bound, sperm can undergo the acrosome reaction, which results in their release from ZP3-beads. (6) Islet-activating protein and 3-quinuclidinyl ben-zilate, two inhibitors of the zona pellucida-induced acrosome reaction, prevent sperm bound to ZP3-beads from undergoing the acrosome reaction. These results confirm and extend previous studies of sperm-egg interaction in mice, and suggest that the solid-phase assay will be useful for both cellular and biochemical analyses of mammalian fertilization.
Isolation and biochemical characterization of septate junctions: Differences between the proteins in smooth and pleated varieties
Authors: Lane Nancy J. , Dilworth Stephen M. 
Published: April 2021

ABSTRACT
Septate junctions are found only in invertebrate tissues, and are almost ubiquitous within them. In arthropods, the two major types are the ‘pleated’ and the ‘smooth’ varieties. Using tissues from different species, including the cockroach Periplaneta americana, procedures have been established for obtaining membrane fractions selectively enriched in septate junctions. The junctions have been identified in pellets of these fractions by both thin sectioning and freeze-fracturing. SDS-PAGE of these membrane fractions reveals two major polypeptide species with apparent molecular weights of 22 000–24 000 and 17000–18000. Consistent differences in these apparent molecular weights are observed between the pleated and smooth varieties of septate junction. These polypeptides are probably integral membrane components, as they remain associated after treatment with high concentrations of urea. Evidence suggests a plane of weakness in the mid-line of the extracellular septal ribbons.
Monoclonal antibody against the centrosome
Authors: Rao Potu N. , Zhao Ji-Ying , Ganju Ramesh K. , Ashorn Cheryl L. 
Published: April 2021

ABSTRACT
A monoclonal antibody, MPM-13, raised against mitotic HeLa cell extracts recognized a perinuclear area in interphase cells and spindle poles in mitotic cells of human, mouse, Chinese hamster and sea urchin. On immunoblots MPM-13 recognized a major protein band at 43×103Mr and a variable minor band at 56 ×103Mr in both mitotic and interphase HeLa cells. These antigens were detectable in a variety of mammalian cells as well as in the unicellular ciliate Tetrahymena. In cells arrested in mitosis by colcemid and stained with MPM-13 by indirect immunofluorescence, numerous fluorescent speckles were seen throughout the cytoplasm. Reversal of colcemid block in Chinese hamster ovary (CHO) cells by washing resulted in the reappearance of a fluorescent patch at the poles of the re-formed spindles. In HeLa cells arrested in mitosis by the microtubule stabilizing drug taxol, MPM-13 stained a large fluorescent patch encircled by dark metaphase chromosomes. This pattern indicated the failure of centrioles to move to the opposite poles in the presence of taxol. These data indicate that the MPM-13 antigens are associated with the colcemid-sensitive pericentriolar material from which microtubules originate but not with the centrioles themselves. It is also clear that these antigens are highly conserved during evolution.
Expression in Escherichia coli of fragments of glial fibrillary acidic protein: Characterization, assembly properties and paracrystal formation
Authors: Quinlan Roy A. , Moir Robert D. , Stewart Murray 
Published: April 2021

ABSTRACT
We have expressed in Escherichia coli a 1258 bp cDNA fragment corresponding to 97% of mouse glial fibrillary acidic protein (GFAP), the principal intermediate filament protein of astrocytes. High levels of expression were obtained, as a fusion protein with 32 residues of the bacteriophage Â ell protein, using the pLcII expression vector system of K. Nagai and H.-C. Thogersen. Although removal of the cH protein fragment by proteolysis using factor X proved difficult, a protein corresponding to most of the cDNA fragment was obtained by cleaving at the endogenous thrombin site near the middle of the N-terminal non-helical domain of GFAP. A shorter 1047 bp fragment, in which the C-terminal non-helical domain of GFAP was deleted, was also produced using oligonucleotide-directed sitespecific mutagenesis of the original cDNA clone. After proteolysis with thrombin, this material gave a fragment that corresponded to the a-helical coiled-coil rod region of the GFAP molecule, together with a portion of the non-helical N-terminal domain. The fragments produced were characterized both biochemically and ultrastruc-turally, and appeared to retain the conformation of native GFAP. Crosslinking showed that all fragments formed molecules containing two chains (‘dimers’) that associated to form four-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 proteolytically prepared rod domains. Remarkably, the fusion protein produced from the entire 1258 bp cDNA fragment and the ell peptide was able to form filaments that closely resembled those produced by native GFAP. However, fragments in which either the cH peptide or the C-terminal nonhelical domain were removed, or in which both were removed, failed to form filaments under standard assembly conditions. Although preliminary in nature, these results suggest that both N- and C-terminal non-helical domains may have a role in intermediate filament formation. Moreover, the fragment corresponding approximately to the GFAP rod formed paracrystals similar to those observed with other coiled-coil proteins. The molecules in these paracrystals were arranged antiparallel with the two molecules in the unit cell, which may correspond to the Jour-chain molecular dimer (tetramer), overlapping by approximately two-thirds of their length.