bluuebunny/crossref_metadata_2025 huggingface dataset:
Positional reorganization in compoundjanuscells ofTetrahymena thermophila
Authors: Frankel Joseph , Nelsen E. Marlo
Published: April 2021

The janus mutations of Tetrahymena thermophila convert the large-scale organization of the dorsal surface of the cell into a mirror-image of the ventral surface, which is characterized by a second, abnormal, oral apparatus and by contractile vacuole pores to the left of the second oral area rather than the usual right. This conversion could be due either to a local change in the response to an unaltered positional system or to a more global reorganization of the system itself, janus homopolar doublets were used to distinguish between these two alternatives. Homopolar doublets can be made by fusing two similarly oriented cells in side-by-side parabiosis. Non-janus homopolar doublets typically possess two sets of normal oral structures with contractile vacuole pores to the right of each of them. In janus doublets, there are up to four sets of oral structures, with the abnormal oral structures located between the two sets of normal oral structures; contractile vacuole pores are situated to the right of the normal oral areas and to the left of the abnormal oral structures.

Non-janus homopolar doublets are known to propagate their compound condition for a number of cell divisions, but also to regulate toward the singlet state through a progressive reduction in number of ciliary rows followed by loss of one of the two sets of major cell surface structures, janus homopolar doublets go through a corresponding regulation. As a consequence, the location of the abnormal oral structures relative to the normal ones is more variable in janus doublets than in janus singlets. Sometimes the abnormal oral structures shift to a position close to their normal counterparts and then the intervening CVP sets disappear. There is evidence for occasional fusion of an abnormal oral area with an adjacent normal oral apparatus, a condition that may be transitional to the singlet state. These observations are inconsistent with the idea of a fixed positional system and strongly suggest a global reorganization of the surface pattern in a manner consistent with predictions of an intercalation model that was first proposed to explain the regulation of non-janus doublets to singlets.
Brachial muscles of dystrophic chick embryos atypically sustain interaction with thoracic nerves
Authors: Butler Jane , Cosmos Ethel
Published: April 2021

Previous analyses of experimental chick embryos of normal lineage demonstrate the inability of brachial muscles to sustain a successful union with foreign nerves derived from a thoracic neural tube segment transplanted to the brachial region at day 2 in ovo (day 2E). The present experiments were performed to determine if mutant chick embryos afflicted with hereditary muscular dystrophy would respond similarly to this experimental manipulation. Using the same criteria applied to our analysis of experimental normal embryos, our results demonstrated that dystrophic brachial muscles were capable of maintaining a compatible union with foreign thoracic nerves throughout the experimental period analysed. Significant muscle growth occurred, intramuscular nerve branches were maintained, motor endplates formed and wing motility was equivalent to that of unoperated dystrophic embryos. Thus, foreign nerves rejected by normal brachial muscles were accepted by brachial muscles of the mutant dystrophic embryo.
The effect of the zone of polarizing activity (ZPA) on the anterior half of the chick wing bud
Authors: Wilson D. J. , Hinchliffe J. R.
Published: April 2021

Removal of the posterior half of the chick wing bud between stages 17-22 results in failure of the anterior distal tissue to survive and differentiate. This observation has been interpreted in terms of a requirement by the anterior half of a factor supplied by the posterior half of the limb containing the zone of polarizing activity (ZPA). This relationship has been tested by grafting ZPA tissue to the posterior surface of the anterior half after posterior half removal. Grafts made proximally on the cut surface did not significantly improve survival and development, nor did the ZPA prevent the expansion of the cell death in the ANZ beyond its normal boundaries into the distal mesenchyme. However, when grafted distally the ZPA inhibited cell death in the apical mesenchyme and caused the anterior mesenchyme to change its normal prospective fate (radius and digit 2). In all these cases, in addition to digit 2, digit 3 and frequently also digit 4 differentiated. The anterior half went on to develop a full set of digits and zeugopod parts in almost 50 % of cases, although no skeleton resulting from this regulation of the anterior half had totally size regulated. These results demonstrate a developmental ‘rescue’ effect by the ZPA, and further support the view that the ZPA has a central and unique function in normal limb bud development, controlling survival and differentiation of the mesenchyme along the anteroposterior axis.
M-phase-promoting factor activation
Authors: Meikrantz William , Schlegel Robert A.
Published: April 2021

Two decades ago, Hartwell and co-workers isolated a number of cell division cycle (cdc) mutants in Saccharomyces cerevisiae on the basis of their arrest at specific, morphologically distinguishable points in the cell cycle. These temperature-sensitive mutants provided the first identification and temporal ordering of genes required for progress through the cell cycle, and permitted the first molecular definition of a cell cycle restriction point, START, passage through which requires the function of the CDC28 gene. Interestingly, CDC28 was found to be required not only for leaving stationary phase and commencing DNA replication, but also for the events of nuclear division (Hartwell et al., 1974; Hartwell and Weinert, 1989; Reed et al., 1985). Similarly, in the fission yeast Schizosaccharomyces pombe, the homologous cdc2 gene was found to be necessary for executing both the G1/S and G2/M transitions (Nurse et al., 1976; Nurse and Bisset, 1981; Beach et al., 1981). CDC28/cdc2, and their homologs in species spanning the plant and animal kingdoms, encode protein kinases of approximately 34 kDa (Hindley and Phear, 1984; Reed et al., 1985; Simanis and Nurse, 1986), referred to hereafter simply as p34. In animal cells, increasingly sophisticated molecular techniques have led to the identification of a family of p34-related genes (Pines and Hunter, 1991), some of which are clearly distinct from CDC28 or cdc2 and may mediate some of the G1/S functions previously attributed exclusively to CDC28/cdc2 (Elledge and Spottswood, 1991; Fang and Newport, 1991; Koff et al., 1991; Lehner and O’Farrell, 1990; Paris et al., 1991; Tsai et al., 1991).
Morphological and immuno-cytochemical characterization of a hetero-spheroid composed of fibroblasts and hepatocytes
Authors: Takezawa Toshiaki , Yamazaki Manabu , Mori Yuichi , Yonaha Tomohide , Yoshizato Katsutoshi
Published: April 2021

A novel method for the preparation of spheroids containing two types of cells (hetero-spheroid) has been successfully developed by utilizing a collagen-conjugated thermo-responsive polymer, poly-N-isopropyl acrylamide (PNIPAAm), as a cell substratum. PNIPAAm solidifies above its lower critical solution temperature (LCST, about 30°C), and instantly dissolves into the culture medium below its LCST. We firstly seeded and cultured human dermal fibroblasts on the substratum up to a confluent state and then seeded rat primary hepatocytes onto the fibroblast monolayer. The hetero-spheroid was prepared by detaching the hepatocyte-attached fibroblast monolayer at a temperature below LCST and culturing it on the non-adhesive substratum. The surface area of the substratum and the seeding population ratio of each cell precisely and reproducibly regulated the size and the cell composition of the resulting hetero-spheroid, respectively. Histological and immuno-cytochemical observations of spheroids revealed characteristic organizations of fibroblasts and hepatocytes within a spheroid because the latter cells expressed albumin for up to at least 3 weeks. TEM study of the hetero-spheroid showed the presence of structures morphologically similar to the Disse’s space and the bile canaliculus, which are features characteristic of liver. These findings suggest that the method described above is useful for making a hetero-spheroid that morphologically and functionally resembles tissues or organs in vivo, i.e. an organoid.
Cytoplasmic accumulation of cyclin B1 in human cells: Association with a detergent-resistant compartment and with the centrosome
Authors: Bailly Eric , Pines Jonathon , Hunter Tony , Bornens Michel
Published: April 2021

Mitotic cyclins are thought to function as key regulatory subunits of the universal M-phase-promoting p34cdc2 protein kinase. Previous immunolocalization studies have demonstrated that a fraction of p34cdc2 undergoes cell cycle-dependent accumulation at the centrosome during G2/M. In order to identify the mitotic cyclins with which this p34cdc2 fraction interacts, we carefully examined the subcellular distribution of both cyclin A and Bl in HeLa cells. We show here that part of cyclin Bl is associated with duplicating centrosomes throughout its accumulation in the cytoplasm and up to metaphase. In contrast cyclin A does not exhibit centrosomal association except at the onset of mitosis, from preprophase up to metaphase. We also present cytological and biochemical evidence that cyclin Bl is preferentially accumulated as a detergent-insoluble protein independently of the state of microtubule assembly and under experimental conditions where most of p34cdc2 is soluble. Interestingly, the electrophoretic pattern of the minor insoluble p34cdc2 fraction was previously shown to be particularly enriched in slow-migrating and presumably hyperphosphorylated isoforms, known to interact specifically with cyclin Bl during interphase. From these results we propose that the interaction of cyclin Bl with the centrosomes and with the cytoplasmic structures is a constitutive feature of the mechanism whereby a fraction of p34cdc2 is recruited and subsequently targeted to the cyclin B-dependent activation pathway.
Protein kinase C isotypes in human erythroleukemia cell proliferation and differentiation
Authors: Hocevar Barbara A. , Morrow Dwight M. , Tykocinski Mark L. , Helds Alan P.
Published: April 2021

The human erythroleukemia (K562) cell line is induced to differentiate into megakaryocytic cells by treatment with the tumor promoter phorbol myristate acetate (PMA). PMA-induced differentiation is characterized by (1) almost complete cessation of cellular proliferation, (2) expression of the megakaryocytic cell surface marker glycoprotein Ilb/IIIa (gpHIa), (3) increased secretion of granulocyte/macrophage-colony stimulating factor (GM-CSF) and (4) increased secretion of interleukin-6 (IL-6). PMA-induced differentiation is dose-dependent with maximal activity seen at 10 nM PMA. In contrast, bryostatin (bryo), a structurally distinct protein kinase C (PKC) activator, fails to induce megakaryocytic differentiation or growth arrest at the concentrations tested (0.01-100 nM). Rather, bryo inhibits PMA-induced growth arrest and megakaryocytic differentiation in a dose-dependent fashion (full inhibition at 100 nM). The divergent biological effects of PMA and bryo correspond to the differential activation and translocation of PKC isotypes in K562 cells. PKC isotype analysis demonstrates that undifferentiated cells express both α and βII PKC but no detectable β I, γ or ∈ PKC.

Treatment of cells with either PMA or bryo leads to rapid translocation of both α and βII PKC from the cytosol to the non-nuclear particulate fraction. However, bryo also induces selective translocation of βII PKC to the nuclear membrane. Nuclear βII PKC is functionally active as evidenced by the time-dependent phosphorylation of lamin B, a previously identified nuclear PKC substrate. These data indicate that the divergent effects of PMA and bryo on erythroleukemia cell proliferation and differentiation correspond to differential activation of βII PKC at the nuclear membrane. Nuclear activation of βII PKC by bryo appears to generate a dominant, proliferative signal that overrides the PMA-induced differentiation signal. Therefore, the α and βII PKC isotypes exhibit distinct translocation and activation profiles during megakaryocytic differentiation and proliferation, indicating that they play distinct roles in these cellular processes.
FAP-1 in pancreatic cancer cells
Authors: Ungefroren Hendrik , Kruse Marie-Luise , Trauzold Anna , Roeschmann Stefanie , Roeder Christian , Arlt Alexander , Henne-Bruns Doris , Kalthoff Holger
Published: April 2021

In this study we investigated the functional role of FAP-1 as a potential inhibitor of CD95 (Fas, APO-1)-mediated apoptosis in pancreatic cancer cells. Stable transfection of the CD95-sensitive, FAP-1-negative cell line Capan-1 with an FAP-1 cDNA resulted in a strongly decreased sensitivity to CD95-induced apoptosis, as measured by DNA fragmentation and caspase-3 activity. Inhibition of cellular protein tyrosine phosphatases with orthovanadate dose-dependently increased CD95-induced apoptosis in CD95-resistant FAP-1-positive Panc89 and Capan-1-FAP-1 cells almost to the level seen in wild-type Capan-1 cells. Blocking the CD95/FAP-1 interaction in Panc89 cells by cytoplasmic microinjection of a synthetic tripeptide mimicking the C terminus of CD95 resulted in a mean 5.5-fold increase in apoptosis compared to cells that received a control peptide. Using confocal laser scanning microscopy we show that in Panc89 cells FAP-1 is mainly associated with the Golgi complex and with peripheral vesicles. FAP-1 displayed enhanced colocalization with CD95 upon CD95 stimulation in the Golgi complex but not in surface-associated vesicles. This correlated with a decrease in plasma membrane staining for CD95 as determined by FACS analysis. Inhibition of Golgi anterograde transport by brefeldin A abolished the anti-CD95-induced colocalization of FAP-1 and CD95 as well as the decrease in cell-surface-associated CD95. Finally, we demonstrate by immunohistochemistry that FAP-1 is strongly expressed in tumor cells from pancreatic carcinoma tissues. Taken together, these results show that FAP-1 can protect pancreatic carcinoma cells from CD95-mediated apoptosis, probably by preventing anti-CD95-induced translocation of CD95 from intracellular stores to the cell surface.
The Ccz1 protein interacts with Ypt7 GTPase during fusion of multiple transport intermediates with the vacuole inS. cerevisiae
Authors: Kucharczyk Róża , Kierzek Andrzej M. , Slonimski P. P. , Rytka Joanna
Published: April 2021

Previously we have shown that the Saccharomyces cerevisiae CCZ1 (YBR131w) gene encodes a protein involved in protein trafficking. Deletion of this gene leads to fragmentation of the vacuole typical of the class B vps mutants. Genetic and biochemical data indicated that Ccz1p is required for fusion of various transport intermediates with the vacuole. Here we present data indicating that CCZ1 is a close partner of the YPT7 gene, which encodes Rab GTPase and is required for fusion of transport vesicles to vacuole and homotypic vacuole fusion. We isolated extragenic suppressors of CCZ1 deletion. All these suppressors belong to one complementation group and correspond to mutated alleles of the YPT7 gene. The mutated residues are located in two Ypt7p domains responsible for guanine binding. These data suggest that Ccz1p and Ypt7p interact physically. Coimmunoprecipitation experiments provide direct evidence that this indeed is the case. A possible mechanism of Ccz1p action is discussed.
Revealing the unseen: the organizer region of the nucleolus
Authors: Biggiogera Marco , Malatesta Manuela , Abolhassani-Dadras Sousan , Amalric François , Rothblum Lawrence I. , Fakan Stanislav
Published: April 2021

We carried out a high-resolution ultrastructural analysis of the nucleolus in mouse P815 cells by combining specific DNA and RNA staining, anti-fibrillarin immunolabeling, contrast enhancement by energy filtering TEM and phosphorus mapping by ESI to visualize nucleic acids. We demonstrated that specifically contrasted DNA, fibrillarin and phosphorus overlap within the nucleolar dense fibrillar component. Moreover, we describe a ‘DNA cloud’ consisting of an inner core of DNA fibers (fibrillar center) and a periphery made of extremely thin fibrils overlapping the anti-fibrillarin immunolabeling (dense fibrillar component). This highly sensitive approach has allowed us to demonstrate, for the first time, the exact distribution of DNA within the decondensed interphase counterpart of the NOR, which includes both the fibrillar center and the dense fibrillar component.
The neuropeptide head activator induces activation and translocation of the growth-factor-regulated Ca2+-permeable channel GRC
Authors: Boels Katrin , Glassmeier Günter , Herrmann Doris , Riedel I. Björn , Hampe Wolfgang , Kojima Itaru , Schwarz Jürgen R. , Schaller H. Chica
Published: April 2021

The neuropeptide head activator stimulates cell proliferation of neuronal precursor and neuroendocrine cells. The mitogenic signaling cascade requires Ca2+ influx for which, as we show in this paper, the growth-factor-regulated Ca2+-permeable cation channel, GRC, is responsible. GRC is a member of the transient receptor potential channel family. In uninduced cells only low amounts of GRC are present on the plasma membrane but, upon stimulation with head activator, GRC translocates from an intracellular compartment to the cell surface. Head activator functions as an inducer of GRC translocation in neuronal and neuroendocrine cells, which express GRC endogenously, and also in COS-7 cells after transfection with GRC. Head activator is no direct ligand for GRC, but its action requires the presence of a receptor coupled to a pertussis-toxin inhibitable G-protein. Heterologously expressed GRC becomes activated by head activator, which results in opening of the channel and Ca2+ influx. SK&F 96365, an inhibitor specific for TRP-like channels, blocks Ca2+ entry and, consequently, translocation of GRC is prevented. Head activator-induced GRC activation and translocation are also inhibited by wortmannin and KN-93, blockers of the phosphatidylinositol 3-kinase and of the Ca2+/calmodulin-dependent kinase, respectively, which implies a role for both kinases in head-activator signaling to GRC.
IP3 receptor function and localization in myotubes: an unexplored Ca2+ signaling pathway in skeletal muscle
Authors: Powell Jeanne A. , Carrasco Maria Angelica , Adams Dany S. , Drouet Beatrice , Rios Juan , Müller Marioly , Estrada Manuel , Jaimovich Enrique
Published: April 2021

We present evidence for an unexplored inositol 1,4,5-trisphosphate-mediated Ca2+ signaling pathway in skeletal muscle. RT-PCR methods confirm expression of all three known isotypes of the inositol trisphosphate receptor in cultured rodent muscle. Confocal microscopy of cultured mouse muscle, doubly labeled for inositol receptor type 1 and proteins of known distribution, reveals that the receptors are localized to the I band of the sarcoplasmic reticulum, and this staining is continuous with staining of the nuclear envelope region. These results suggest that the receptors are positioned to mediate a slowly propagating Ca2+ wave that follows the fast Ca2+ transient upon K+ depolarization. This slow wave, imaged using fluo-3, resulted in an increase in nucleoplasmic Ca2+ lasting tens of seconds, but not contraction; the slow wave was blocked by both the inositol trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and the phospholipase C inhibitor U-73122. To test the hypothesis that these slow Ca2+ signals are involved in signal cascades leading to regulation of gene expression, we assayed for early effects of K+ depolarization on mitogen-activated protein kinases, specifically extracellular-signal related kinases 1 and 2 and the transcription factor cAMP response element-binding protein (CREB). Within 30-60 seconds following depolarization, phosphorylation of both the kinases and CREB was evident and could be inhibited by 2-aminoethoxydiphenyl borate. These results suggest a signaling system mediated by Ca2+ and inositol trisphosphate that could regulate gene expression in muscle cells.
FIGQY phosphorylation defines discrete populations of L1 cell adhesion molecules at sites of cell-cell contact and in migrating neurons
Authors: Jenkins Scott M. , Kizhatil Krishnakumar , Kramarcy Neal R. , Sen Anindita , Sealock Robert , Bennett Vann
Published: April 2021

Phosphorylation of neurofascin, a member of the L1 family of cell adhesion molecules (L1 CAMs), at the conserved FIGQY-tyrosine abolishes the ankyrin-neurofascin interaction. This study provides the first evidence, in Drosophila melanogaster and vertebrates, for the physiological occurrence of FIGQY phosphorylation in L1 family members. FIGQY tyrosine phosphorylation is localized at specialized cell junctions, including paranodes of sciatic nerve, neuromuscular junctions of adult rats and Drosophila embryos, epidermal muscle attachment sites of Drosophila, and adherens junctions of developing epithelial cells of rat and Drosophila. In addition, FIGQY-phosphorylated L1 CAMs are abundantly expressed in regions of neuronal migration and axon extension, including the embryonic cortex, the neonatal cerebellum and the rostral migratory stream, a region of continued neurogenesis and migration throughout adulthood in the rat. Based on our results, physiological FIGQY-tyrosine phosphorylation of the L1 family likely regulates adhesion molecule-ankyrin interactions establishing ankyrin-free and ankyrin-containing microdomains and participates in an ankyrin-independent intracellular signaling pathway at specialized sites of intercellular contact in epithelial and nervous tissue.
mARVCF cellular localisation and binding to cadherins is influenced by the cellular context but not by alternative splicing
Authors: Waibler Zoe , Schäfer Annette , Starzinski-Powitz Anna
Published: April 2021

ARVCF, a member of the catenin family, is thought to contribute to the morphoregulatory function of the cadherin-catenin complex. Recently, we reported the isolation and characterisation of murine ARVCF (mARVCF), particularly its interaction with M-cadherin. Here, we describe the identification of novel mARVCF isoforms that arise by alternative splicing. At the N-terminus, alternative splicing results in the inclusion or omission of a coiled-coil region probably important for protein-protein interactions. At the C-terminus, four isoforms also differ by domains potentially important for selective protein-protein interaction. The eight putative mARVCF isoforms were expressed as EGFP-fusion proteins in six different cell lines that exhibit a distinct pattern of cadherins. Apparently, binding of the mARVCF isoforms to M-, N-, or E-cadherin is generally unaffected by their altered N- and C-termini, as revealed by the MOM recruitment assay. However, mARVCF isoforms reproducibly exhibit differential localisation in distinct cellular environments. For example, mARVCF isoforms are unable to colocalise with N-cadherin in EJ28 carcinoma cells but do so in HeLa cells. Our results suggest that the subcellular localisation of mARVCF may be determined not only by the presence or absence of an appropriate interaction partner, in this case cadherins, but also by the cellular context.
Asymmetric cell division in fucoid algae: a role for cortical adhesions in alignment of the mitotic apparatus
Authors: Bisgrove Sherryl R. , Kropf Darryl L.
Published: April 2021

The first cell division in zygotes of the fucoid brown alga Pelvetia compressa is asymmetric and we are interested in the mechanism controlling the alignment of this division. Since the division plane bisects the mitotic apparatus, we investigated the timing and mechanism of spindle alignments. Centrosomes, which give rise to spindle poles, aligned with the growth axis in two phases – a premetaphase rotation of the nucleus and centrosomes followed by a postmetaphase alignment that coincided with the separation of the mitotic spindle poles during anaphase and telophase. The roles of the cytoskeleton and cell cortex in the two phases of alignment were analyzed by treatment with pharmacological agents. Treatments that disrupted cytoskeleton or perturbed cortical adhesions inhibited pre-metaphase alignment and we propose that this rotational alignment is effected by microtubules anchored at cortical adhesion sites. Postmetaphase alignment was not affected by any of the treatments tested, and may be dependent on asymmetric cell morphology.
Adaptive concentrations of hydrogen peroxide suppress cell death by blocking the activation of SAPK/JNK pathway
Authors: Kim Do Kyun , Cho Eun Sook , Seong Je Kyung , Um Hong-Duck
Published: April 2021

Low levels of H2O2 can induce cellular resistance to subsequent higher levels of H2O2. By using human U937 leukemia cells, it was previously shown that such an adaptive response can be induced without increasing the cellular capacity to degrade H2O2, thus conferring on the cells a cross-resistance to other stimuli such as serum withdrawal and C2-ceramide. In this study, it was found that stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) acts as a common mediator of the cell death induced by high H2O2 concentrations, serum withdrawal and C2-ceramide. Although SAPK/JNK activation by H2O2 was mediated by two upstream mitogen-activated protein kinase (MAPK) kinases MKK4 and MKK7, only MKK7 played such a role in serum withdrawal and C2-ceramide. Interestingly, all these lethal stimuli failed to activate SAPK/JNK and its upstream kinases in the cells that were pretreated with low adaptive concentrations of H2O2. By contrast, the phosphorylation levels of extracellular signal-regulated kinase and p38 MAPK were not significantly influenced by this H2O2 pretreatment. Inducing the SAPK/JNK-suppressing effect of H2O2 required a time lag, which correlated with the time lag required for the induction of the adaptive response. Overall, the results suggest that H2O2 adaptation confers on cells a resistance to multiple stimuli by specifically blocking their ability to activate the SAPK/JNK pathways.
Smad regulation in TGF-β signal transduction
Authors: Moustakas Aristidis , Souchelnytskyi Serhiy , Heldin Carl-Henrik
Published: April 2021

Smad proteins transduce signals from transforming growth factor-β (TGF-β) superfamily ligands that regulate cell proliferation, differentiation and death through activation of receptor serine/threonine kinases. Phosphorylation of receptor-activated Smads (R-Smads) leads to formation of complexes with the common mediator Smad (Co-Smad), which are imported to the nucleus. Nuclear Smad oligomers bind to DNA and associate with transcription factors to regulate expression of target genes. Alternatively, nuclear R-Smads associate with ubiquitin ligases and promote degradation of transcriptional repressors, thus facilitating target gene regulation by TGF-β. Smads themselves can also become ubiquitinated and are degraded by proteasomes. Finally, the inhibitory Smads (I-Smads) block phosphorylation of R-Smads by the receptors and promote ubiquitination and degradation of receptor complexes, thus inhibiting signalling.
The nuclear localization domain of the MEF2 family of transcription factors shows member-specific features and mediates the nuclear import of histone deacetylase 4
Authors: Borghi Serena , Molinari Susanna , Razzini Giorgia , Parise Flavia , Battini Renata , Ferrari Stefano
Published: April 2021

Targeting of myocyte enhancer binding factor 2 (MEF2) proteins to the nucleus depends on a C-terminal bipartite nuclear localization signal (NLS). By expression of green fluorescent protein (GFP)/MEF2 fusion proteins in transfected myoblasts, we show that MEF2C contains an additional 13 amino acids domain, located immediately upstream of the NLS, which contributes to its nuclear retention. We also show that the NLS present in MEF2 proteins is required for efficient nuclear localization of histone deacetylase 4 (HDAC4). In muscle cells, transfected HDAC4 is largely cytoplasmic or, to a lesser extent, pancellular. Co-transfection of either MEF2A or MEF2C causes HDAC4 to accumulate in the nucleus in association with MEF2. This effect strongly depends on MEF2 NLS; it also requires the specific interaction of HDAC4 with MEF2, since the isolated NLS is not sufficient for targeting HDAC4 to the nucleus and other nuclear proteins, such as NF-Y, cannot substitute MEF2. Therefore, we demonstrate that HDAC4, different from HDAC5, is mainly a cytoplasmic resident protein, requiring a trans-acting NLS for nuclear localization. The physiological implications of MEF2 carrying its own inhibitor to the nucleus are discussed.
Interaction of the endoplasmic reticulum α1,2-mannosidase Mns1p with Rer1p using the split-ubiquitin system
Authors: Massaad Michel J. , Herscovics Annette
Published: April 2021

The α1,2-mannosidase Mns1p involved in the N-glycosidic pathway in Saccharomyces cerevisiae is a type II membrane protein of the endoplasmic reticulum. The localization of Mns1p depends on retrieval from the Golgi through a mechanism that involves Rer1p. A chimera consisting of the transmembrane domain of Mns1p fused to the catalytic domain of the Golgi α1,2-mannosyltransferase Kre2p was localized in the endoplasmic reticulum of Δpep4 cells and in the vacuoles of rer1/Δpep4 by indirect immunofluorescence. The split-ubiquitin system was used to determine if there is an interaction between Mns1p and Rer1p in vivo. Co-expression of NubG-Mns1p and Rer1p-Cub-protein A-lexA-VP16 in L40 yeast cells resulted in cleavage of the reporter molecule, protein A-lexA-VP16, detected by western blot analysis and by expression of β-galactosidase activity. Sec12p, another endoplasmic reticulum protein that depends on Rer1p for its localization, also interacted with Rer1p using the split-ubiquitin assay, whereas the endoplasmic reticulum protein Ost1p showed no interaction. A weak interaction was observed between Alg5p and Rer1p. These results demonstrate that the transmembrane domain of Mns1p is sufficient for Rer1p-dependent endoplasmic reticulum localization and that Mns1p and Rer1p interact. Furthermore, the split-ubiquitin system demonstrates that the C-terminal of Rer1p is in the cytosol.
Effects of cytochalasins on the initial aggregation in vitro of embryonic chick cells
Authors: Appleton J. C. , Kemp R. B.
Published: April 2021

The initial aggregation of trypsin-dissociated cells from the skeletal muscle tissue of 9-day-old chick embryos in the presence of cytochalasins A and B was studied in order to discover the effects of these agents on contact and adhesion.

Cytochalasin B (3 μg/ml) had a negligible effect on the rate of aggregation of cells over an 8-h period, but cytochalasin A at concentrations between 3 and 20 μg/ml markedly inhibited aggregation. Both agents altered the shape and size of aggregates and caused cells at their periphery to appear more spherical.

The oxygen uptake of the treated cells was not noticeably different from that of the controls, despite the severe inhibition of isotopic carbon dioxide evolution. The effect of cytochalasin B on cell aggregation was reversible and although the cytochalasin A effect could not be abolished on return to medium free of A, the unaltered oxygen consumption was taken as an indication that permanent cellular injury did not occur.

The effect of the cytochalasins on aggregate structure was interpreted on the basis of arrested cellular motility, but the singular inhibition by cytochalasin A of the rate of aggregation must await final confirmation of its site of action.
Fine-structural changes in a lepidopteran nervous system during metamorphosis
Authors: McLaughlin Barbara J.
Published: April 2021

The fine structure of the metamorphosing abdominal nerve cord of Manduca sexta has been studied. In fifth instar larvae, the connectives are ensheathed by a complex, thickened neural lamella. The underlying perineurium at this stage consists of 2 layers. The outer layer consists of interdigitating type I cells which are attached to the overlying neural lamella by hemi-desmosomes, and to each other by occasional gap and tight junctions which persist throughout development. They are attached by desmosomes to a thin underlying type II cell layer, which is joined by gap and tight junctions and which has desmosomal attachments with the underlying glial membranes. The larval axons are surrounded by multiple glial wrappings containing bundles of microtubules. During the first week after larval-pupal ecdysis, the neural lamella degenerates and is phagocytosed by invading haemocytes. The underlying perineurial I cells gradually become hypertrophied and vacuolated. At the same time the type II layer, which does not increase in size, appears to be composed of either one or two cells which form a continuous ‘bracelet’ around each connective. The cellular bracelet is joined at one or two places by extensive gap, tight and septate junctions, and gap junctions are also seen along its perineurial I and glial borders. The underlying axons are embedded in vast amounts of glial cytoplasm containing relatively few microtubules. During the second week after larval-pupal ecdysis, the neural lamella is reformed and the perineurium flattens again. Type I and II cell junctions remain as described in earlier stages. Before adult emergence, the axons are again wrapped by glial cells rich in microtubules.
Variation in rdna redundancy level and nucleolar organizer length in normal and variant lines of the mexican axolotl
Authors: Sinclair J. H. , Carroll Carole R. , Humphrey R. R.
Published: April 2021

The level of redundancy of ribosomal genes, and the relationship of this level to nucleolar formation at different stages of embryonic development, have been examined in the Mexican axolotl, Ambystoma mexicanum. Individuals from 4 inbred stocks were examined, as well as descendants from 2 nucleolar variants which, in the heterozygous condition, are distinguished by exceptionally small nucleoli. Ribosomal RNA-DNA hybridization assays show that one of the 4 wild type lines has only about one-third as much ribosomal DNA (rDNA) as the other three. One of the nucleolar variants has the same level of rDNA as the larger wild-type level; the other variant has the same amount as the smaller ribosomal genome line. Both original nucleolar variants arose as F1, progeny of crosses between a large rDNA genome line and the small genome line. Cytological examination of pregastrula stage embryos from wild type and nucleolar variant lines show that the lengths of the nucleolar organizer regions (NOR) and the sizes of nucleoli formed, are directly correlated with the amount of rDNA present at the nucleolar locus. During gastrulation of the nucleolar variants, however, a transition appears to take place and the amount of rDNA ceases to be the determining factor in nucleolar size. After late gastrula, heterozygous progeny resulting from crosses of either large rDNA genome or small rDNA genome wild type individuals with either nucleolar variant line, have a small and a large nucleolus. The factor or factors associated with this apparent lack of competitive ability of the variant NOR, when opposed to a normal NOR, are unknown. It might be suggested that since the chromosomal alterations which produced the nucleolar variants in both cases eliminated the gene determining the dark colour pattern, they could at the same time have eliminated other genetic material.
The properties and subcellular localization of acid phosphatases in the colourless alga, Polytomella caeca
Authors: Cooper Rosemary A. , Bowen I. D. , Lloyd D.
Published: April 2021

The acid p-nitrophenyl phosphatase of homogenates of Polytomella caeca is a latent acid hydrolase, which is partially inhibited by NaF. Its distribution profile in density gradients (which is similar to that of naphthyl AS-TR phosphatase) suggests that this enzyme is partially lysosomal in location. Cytochemical evidence for the localization of acid phosphatases in fine subcellular structures is presented. Naphthyl AS-TR phosphatase is localized in vacuoles, points of focal degradation, Golgi bodies and dispersed throughout the cytosol. β-Glycero- phosphatase is confined to large vacuoles and the cytosol. The nature of acid phosphatase- containing organelles in P. caeca is discussed in view of the inability to detect eleven other latent acid hydrolases in cell-free homogenates.
An electron-microscope study of the intranucleolar chromatin in root meristematic cells ofAllium cepa
Authors: Chouinard L. A.
Published: April 2021

The various states of condensation and configuration of the chromatin material, contained inside the lacunar regions of the interphase nucleolus in Allium cepa, have been investigated by means of conventional electron-microscope techniques. The observations reveal that in a number of lacunar profiles, the chromatin material in question appears in an extended state only; in other lacunar profiles of the same or different nucleoli, the chromatin material is present both in an extended and a condensed condition. Moreover, in some lacunar profiles, a single mass of chromatin in a condensed state is observed; in others, several discrete and often seemingly interconnected masses of condensed chromatin are visualized.

An attempt is made to interpret these morphological findings in the light of current knowledge concerning the structural relationship of the nucleolar organizing segment of the nucleolar chromosome with the interphase nucleolus in plant cells. The relevant observational evidence would be consistent with the view that the chromatin-containing lacunar regions of the interphase nucleolus in Allium cepa correspond, in fact, to cross or oblique sections of a meandering channel through which the nucleolar organizing segment of the nucleolar chromosome passes. Assuming the applicability to intranucleolar chromatin of the general concept of condensed-inactive versus extended-active chromatin, it is hypothesized that the various states of condensation of intralacunar chromatin merely reflect variations in the functional activity of the nucleolar organizing segment during the interphase cell cycle in the species investigated. With regard to variations in the configurational state of the intralacunar condensed chromatin, it is postulated that they are the cytological expression of the gradual process of coiling or folding upon itself of the nucleolar organizing segment during late interphase and in preparation for the next mitosis.
Modification of sporulation in yeast strains with two-spored asci (Saccharomyces, ascomycetes)
Authors: Moens P. B.
Published: April 2021

Electron-microscope observations indicate that the single nuclear division of sporulating yeasts which form 2-spored asci resembles, in several aspects, the nuclear behaviour during sporulation of the more common 4-spored yeasts. Specifically the parental nucleolus remains behind in the ascus cytoplasm, a characteristic nucleolus-associatcd spherical structure is formed, and the nucleus divides by budding rather than by elongation and medial constriction.
Quantitative evaluation of cell orientation in culture
Authors: Margolis L. B. , Samoilov V. I. , Vasiliev Ju. M. , Gelfand I. M.
Published: April 2021

A quantitative method to assess mutual orientation of cells in cultures on a substrate includes the following operations: (1) the cellular groups to be evaluated are chosen; (2) position of the long axis for each nucleus of the group is determined ; (3) the axis OX is arbitrary chosen for every group and the angles αi between the long axis of every nucleus i and the axis OX are measured. Every nucleus i corresponds to a vector of unit length ei with the angle 2α.

D, the mean of the vectors ei for every cell group is calculated. This value of D is compared with a set of values of D computed according to a model of mutual orientation studied in a simulation experiment. In this model the group of n vectors consists of a fraction of Kn parallel vectors (o < K < 1) and of (1 —K)n randomly oriented vectors. K corresponding to the computed D which is equal to the experimental value of D is considered as an index of orientation for the group. Contact orientation with respect to the relief of the substrate may be evaluated as a root mean square deviation σ0 of the angles between the long axes of cell nuclei and the direction of relief. Examples of the measurements of K and σ0 in cell cultures are given.
Reversion in expression of hypoxanthine-guanine phosphoribosyl transferase following cell hybridization
Authors: Bakay B. , Nyhan W. L. , Croce C. M. , Koprowski H.
Published: April 2021

Hybridization of mutant cell lines deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C.: 2.4.2.8) from a variety of established rodent sources with HGPRT+ human cells yielded progeny cells which grew in selective medium containing hypoxanthine, aminopterin and thymidine (HAT). The same result was obtained when the human cell used was an HGPRT- transformed line derived from a patient with the Lesch-Nyhan syndrome. Electrophoretic analysis indicated that all HAT-resistant progeny clones contained an active HGPRT enzyme which was indistinguishable from the wild type enzyme of the corresponding normal rodent cells. In contrast, no HAT-resistant cells have been obtained when the same HGPRT- rodent cells were subjected to fusion processes in the absence of human cells or when they were fused with similarly derived HGPRT- mutant cells of other rodents. Reversion in expression of the rodent gene for HGPRT was detected in clones which retained one or more human chromosomes and in clones which contained no detectable human chromosomal material. The observed re-expression of rodent HGPRT in HAT-resistant clones suggests that HGPRT+ as well as HGPRT- human cells contributed a factor which determined the expression of respective rodent structural genes for HGPRT. In contrast, HGPRT- rodent cells were unable to induce the synthesis of normal HGPRT in the cells derived from the patient with the Lesch-Nyhan syndrome.
Ultrastructural changes in chloroplasts resulting from fluctuations in nacl concentration: freeze-fracture of thylakoid membranes in Dunaliella salina
Authors: Pfeifhofer A. O. , Belton J. C.
Published: April 2021

The structure of chloroplasts isolated from Dunaliella salina has been studied with respect to changing concentrations of sodium chloride in the culture medium. Freeze-fracture replicas and thin sections of intact chloroplasts do not exhibit any noticeable changes in structure at concentrations ranging between 3·5 and 25 % NaCl. Chloroplasts isolated from algal cells that have been acclimatized to the higher salt concentration show a change in the thylakoid membranes. The thylakoid membranes appear compressed over a major portion of the membrane surface, with only the end of the thylakoid membranes unappressed. The number of particles per unit area on the B face is also altered by the salt concentration. The chloroplasts acclimatized to 25 % NaCl have about 3 times the number of particles per unit area on a B face of endmembranes as on a comparable face of thylakoid membranes acclimatized to low (3·5 % NaCl) salt concentration. These morphological changes can be reversed if the chloroplasts acclimatized to high or low salt concentrations are returned to a medium of different salt concentration prior to freeze-fracturing.
Characterization of an sv40-transformed 3t3 cell line expressing an unusual phenotype
Authors: Thompson J. E. , Elligsen J. D. , Frey H. E.
Published: April 2021

A transformed variant derived as a clone from normal 3T3 cells infected with simian virus 40 (SV40) has been found to possess a phenotype intermediate between that of normal cells and that characteristic of the transformed state, yet cells of the variant still test positively for the SV40-specific nuclear T-antigen. The variant exercises growth control, although not as stringently as do normal cells. Its cell size more closely resembles that of normal cells than of transformed cells. The variant also exhibits levels of spontaneous agglutination that are in line with those characteristic of the normal cells from which it was derived, and far higher than corresponding values for cells exhibiting the fully transformed phenotype. Plasma membranes of variant cells more closely resemble those of transformed cells than of normal cells as estimated by polyacrylamide gel electrophoresis. Perhaps the most distinguishing characteristic of the transformed variant is its complete immunity to agglutination by concanavalin A (Con A), even at concentrations of the lectin as high as 500 μg/ml. Moreover, trypsinization does not render variant cells as agglutinable in the presence of Con A as are untreated fully transformed cells. By contrast the variant displays a low tolerance to Con A toxicity, as monitored by ability to grow after treatment with the lectin, and on this count resembles transformed cells. Moreover a survey of several normal cell lines has revealed that even they do not consistently show resistance to Con A toxicity. These observations indicate that Con A-mediated agglutination and inability to grow after treatment with Con A are quite independent and do not bear a cause and effect relationship.
Mode of action of amphotericin b on chick embryo fibroblasts and on mouse ehrlich tumour cells: a cytological and cytochemical analysis
Authors: Paermentier F. De , Bassleer R. , Lepoint A. , Desaive Cl. , Goessens G. , Lhoest-Gauthier M. -P.
Published: April 2021

Chick embryo fibroblasts cultivated in vitro and Ehrlich ascites tumour cells (in vivo or in vitro) have been treated with amphotericin B. Cell multiplication is strongly inhibited. Large clear zones appear in the fibroblast nucleoli (phase-contrast and electron-microscope observations). Many treated fibroblasts and tumour cells have a high DNA content (pre-mitotic or polyploid level; measurements by cytophotometry). However, the RNA content (cytophotometry) and the total protein content (cytophotometry and micro-interferometry) are relatively low in the tumour cells. As shown by autoradiography, DNA synthesis is active but RNA synthesis and, in some cases, protein synthesis are inhibited. Due to this unbalanced growth, the cells cannot divide.
Electron-microscope observations of mitosis and cytokinesis in multinucleate protoplasts of soybean
Authors: Fowke L. C. , Bech-Hansen C. W. , Gamborg O. L. , Constabel F.
Published: April 2021

Multinucleate soybean protoplasts produced by spontaneous fusion during enzyme digestion of the cell wall initiated cell division after approximately 40 h in culture. The structure of these protoplasts during mitosis and cytokinesis was studied with both light and electron microscopes. Most nuclei did not fuse but divided synchronously. Interphase nuclei were commonly connected by short narrow nuclear bridges. At prophase and metaphase the nuclei appeared typical of those in most higher plants; technical difficulties prevented an adequate examination of protoplasts at anaphase. Telophase was characterized by cytokinesis involving phragmoplast and cell plate formation; however, complete partitioning of the cytoplasm by cell plates was not observed. Numerous coated vesicles were present near to or continuous with the cell plate and plasmalemma. The presence of a few dividing protoplasts with at least double the normal chromosome number suggests that some nuclear fusion occurred prior to mitosis. Very little cell wall material was detected at the margin of the dividing protoplasts.