bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of intracellular calcium in protein secretion in Dictyostelium discoideum
Authors: Coukell M. B. , Cameron A. M. , Adames N. R.
Published: April 2021

We reported previously that Ca2+ depletion of Dic tyostelium discoideum cells severely inhibits extracellular cyclic nucleotide phosphodiesterase (PD) synthesis at a post-transcriptional step. In this study, further experiments were performed to learn more about the nature of this phenomenon. Examination of the polysomal distribution of PD transcripts in control cells and in cells depleted of Ca 2+ by incubation with EGTA and A23187 (EA) suggested that inhibition of PD production does not involve translational control. Kinetic analysis of this inhibitory process revealed that soluble, intracellular PD activity, synthesized from either the 2.4 or 1.9 kb PD mRNA, decreased very rapidly upon addition of EA. Furthermore, this decrease in activity was accompanied by the preferential loss of PD-related polypeptides, indicating a proteolytic event. EA-induced PD degradation required cellular energy and concomitant protein synthesis but was unaffected by most of the lysosomotropic agents tested. Therefore, PD proteolysis might not occur in the lysosome. In cell fractionation experiments, the EA-sensitive, intracellular PD activity comigrated with a rough ER marker in Percoll/KCl gradients. In addition to its effect on the PD, EA were also observed to inhibit production and rapidly lower the intracellular levels of another secreted glycoprotein, the PD inhibitor. Together, these results suggest that depletion of some intracellular Ca2+ store(s) in Dictyostelium, possibly the ER, disrupts the normal function of the secretory pathway, resulting in selective degradation of certain proteins.
Inhibition by kinase inhibitors of the development and the disappearance of the preprophase band of microtubules in tobacco BY-2 cells
Authors: Katsuta Junko , Shibaoka Hiroh
Published: April 2021

The effects of two kinase inhibitors, staurosporine and K-252a, on the development and the disappearance of the preprophase band of microtubules (PPB) were studied in tobacco BY-2 cells, the cell cycle of which had been synchronized. Treatment of cells at late G2 phase or at prophase with 20 M staurosporine or 2 M K-252a for 2 or 3 h caused a decrease in the rate of development of PPBs and inhibited the disappearance of PPBs once they had developed, with the resultant accumulation of cells with a PPB.

Cortical microtubules (MTs) or PPBs on protoplast ghosts, which were prepared from BY-2 cells in late G2 phase or at prophase, disappeared on treatment of the ghosts with ATP or ATP S but not on treatment with AMP-PNP. The effect of ATP on the disappearance of MTs on the ghosts was suppressed by 20 M staurosporine. Although 2 M K-252a applied to the culture medium suppressed the anticipated disappearance of PPBs, it did not suppress the disappearance of MTs on the ghosts that was caused by ATP. We propose tentatively that a kinase sensitive to staurosporine but not to K-252a is directly involved in the disappearance of MTs and that a kinase that is sensitive to K-252a is indirectly involved in this process.

The disappearance of MTs on protoplast ghost that was induced by ATP did not occur in the presence of 20 μM taxol.
Variant choriocarcinoma (BeWo) cells that differ in adhesion and migration on fibronectin display conserved patterns of integrin expression
Authors: Aplin John D. , Sattar Abdul , Mould A. Paul
Published: April 2021

Starting from the BeWo choriocarcinoma cell line, two stable variant cell lines (epi and lc) were isolated. Epi cells displayed an epithelioid colony morphology while lc were fibroblastoid. lc cells attached and spread on fibronectin-coated surfaces at significantly lower density of fibronectin than epi or the parent cell line. lc also migrated more efficiently to fibronectin in a trans-filter assay than either epi or parent cells. Integrin expression by the cell lines was investigated by flow cytometry and immunoprecipitation from surface-labelled cells with a panel of subunit-specific antibodies. Integrins α2β1, α5β1, αvβ1 and α6β4 were detected in each case, and levels of expression were identical in the two variant lines. Anti-functional antibodies were used to probe the role of integrins in fibronectin- and vitronectin-medi-ated adhesion. Complete inhibition of adhesion to fibronectin was observed with anti-β1 antibody, and par-tial inhibition with anti-α5, suggesting that integrin α5β1 is mainly responsible for the interaction. Adhesion to vitronectin was inhibitable using anti-αv and anti-β1 antibodies, suggesting that integrin αvβ1 is active in these cells as a vitronectin receptor. There was a correlation between the altered morphology of the variant cells and alterations in the distribution of integrin α6β4 and laminin in monolayer cultures. The results support the idea that fibronectin may mediate the migratory behaviour of extravillous trophoblast in vivo. Switch to a more migratory phenotype may be mediated by the selective activation of integrins and altered interaction with basement membrane.
Stabilization of intercellular contacts in MDCK cells during Ca2+ deprivation: Selective effects of monocarboxylic acids on desmosomes
Authors: Bogner P. , Skehan P. , Kenney S. , Sainz E. , Akeson M.A. , Friedman S. J.
Published: April 2021

Short-chain monocarboxylic acids (MCAs) selectively protect desmosomal junctions of MDCK cells from disruption by chelating agents and low calcium medium. This effect occurs in the millimolar concentration range and increases inversely with carbon chain length (formate > acetate = propionate > butyrate > isobutyrate > isovalerate). The relative activity of MCAs does not correlate with their overall hydrophobicity or ability to chelate ions, or their effectiveness in lowering cytosolic pH. It exhibits chemical specificity and is dependent upon postconfluency culture age. MCAs also inhibit cell rounding produced by low concentrations of aminocarboxylate-chelating agents. Their effect on cell rounding, but not on desmosomes, can be antagonized by okadaic acid. The possibility is discussed that MCAs may produce their effects by binding specifically to protein(s) associated with the desmosome of mature, fully polarized MDCK monolayers.
Hemidesmosomes in the epithelial cell line 804G: Their fate during wound closure, mitosis and drug induced reorganization of the cytoskeleton
Authors: Riddelle K.S. , Hopkinson S.B. , Jones J.C.R.
Published: April 2021

Recently, we identified a novel epithelial cell line, 804G, derived from rat bladder, which readily forms hemidesmosomes in vitro. One of the major structural components of the plaques of 804G cell hemidesmo-somes is a 230 kDa antigen recognized by autoantibod-ies in the sera of patients with bullous pemphigoid (BP). An additional polypeptide of 180 kDa also localizes to the hemidesmosome plaque of 804G cells as determined by immunoelectron microscopy. Using confocal fluores-cence/phase microscopy, we have employed both 230 kDa and 180 kDa antibody probes to monitor the fate of hemidesmosomes following closure of in vitro wounds, during mitosis, and following drug induced disruption of the cytoskeleton. The punctate cell-substratum asso-ciated staining generated by the hemidesmosomal anti-bodies in stationary unwounded 804G cell cultures is greatly diminished or even lost in cells which enter wound sites, presumably in response to enhanced cell motility. Few, if any, hemidesmosomes are observed at the ultrastructural level in cells which have migrated into the wound area. However, as closure of the wound becomes complete, staining along the substratum attached surface of cells returns. During mitosis, there is no obvious loss of hemidesmosomal antigens along the basal surface of 804G cells, and formed hemidesmo-somes can be observed in mitotic cells at the ultrastructural level. In 804G cells treated with colchicine, the typical subnuclear pattern of distribution of hemidesmosomal antigens is unaffected. In contrast, following treatment of 804G cells with cytochalasin D, hemidesmosomal antigens become concentrated at the cell periphery and no longer appear in the subnuclear region. Furthermore, formed hemidesmosomes are observed at the cell periphery of cytochalasin D-treated cells by electron microscopy. We suggest that hemidesmosomal plaques are mobile within the plasma membrane. We speculate that hemidesmosomal interactions with extracellular ligands are dynamic and we discuss a possible mechanism by which cytochalasin D induces reorganization of hemidesmosomes along the basal surface of 804G cells.
Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro
Authors: Arciniegas E. , Sutton A. B. , Allen T. D. , Schor A. M.
Published: April 2021

α-Smooth muscle actin is considered a reliable marker for distinguishing between arterial smooth muscle and endothelial cells. Several authors have reported heterogeneity in the expression of this actin isoform in atherosclerotic lesions. Such heterogeneity appears to result from the presence of different smooth muscle cell phenotypes (contractile and synthetic) in these lesions. In the present study, we show that bovine aortic endothelial cells, which are characterised by the presence of Factor VIII-related antigen (FVIII) and by the absence of αsmooth muscle actin (α-SM actin) may be induced to express the latter when exposed to TGF-β1. FVIII was detected by immunofluorescence, α-SM actin was detected by immunofluorescence and immunoblotting. The number of cells expressing α-SM actin increased with time of incubation with TGF-β1, and this increase occurred concomitantly with a decrease in the expression of FVIII. Double immunofluorescence demonstrated the presence of cells that expressed both FVIII and α-SM actin after 5 days of incubation with TGF-β1. With longer incubation times (10-20 days) the loss of FVIII expression was complete and over 90% of the cells expressed α-SM actin.

Ultrastructurally, cells in control cultures showed the typical features of endothelial cells. In the TGF-β1-treated cultures, cells which appeared indistinguishable from contractile and synthetic smooth muscle cells were observed. Withdrawal of TGF-β1 after 10 days incubation resulted in the re-appearance of polygonal cells which were FVIII-positive and α-SM actin-negative. Other cells in the same cultures, however, remained ragged in morphology, FVIII-negative and α-SM actinpositive even after 20 days in control medium. This indicates that the inductive effect of TGF-β1 is partly reversible after 10 days incubation. Such reversibility was no longer apparent after 20 days incubation with TGF-β1.

Our results demonstrate that TGF-β1 induces the differentiation of aortic endothelial cells into a smooth muscle-like phenotype and suggest a novel role for TGF-β1 in atherogenesis.
Porcine smooth muscle cell-conditioned medium stimulates LDL receptor activity in hep G2 cells
Authors: Moorby C. D. , Gherardi E. , Riddell D. , Bowyer D. E.
Published: April 2021

Paracrine factors may modulate low density lipoprotein (LDL) receptor activity in hepatocytes. To study this the effect of conditioned medium prepared from a range of cell types on the binding and internalisation of 125I-LDL in Hep G2 cells was studied. Seven of the fourteen con-ditioned media tested, including those from P388D1, U937, porcine smooth muscle (Pc SMC) Swiss 3T3, STO, =48 and MDCK cells, were found to increase the bind-ing and internalisation of 125I-LDL at 37°C by Hep G2 cells (P < 0.01). The largest increase in LDL receptor activity was produced by conditioned medium from Pc SMC cells and was, therefore, selected for further analysis. The Pc SMC-conditioned medium increased LDL receptor number in Hep G2 cells by three-fold but had no effect on LDL receptor activity in human skin fibrob-lasts. DNA synthesis and cholesterol synthesis by Hep G2 cells were inhibited by Pc SMC-conditioned medium. Preliminary characterisation of the Pc SMC-derived factor(s) suggests that it is a protein(s) of low relative molecular mass.
Axonal growth on astrocytes is not inhibited by oligodendrocytes
Authors: Fawcett James W. , Fersht N. , Housden L. , Schachner M. , Pesheva P.
Published: April 2021

Axon growth in vitro may be inhibited by contact with oligodendrocytes, but most axons grow readily on the surface of astrocyte monolayers. Since both cell types are in close contact with one another in the damaged nervous system, we have examined the growth of axons on cultures which contain both astrocytes and oligo-dendrocytes. Cultures derived from neonatal rat fore-brain develop with a monolayer of large flat astrocytes attached to the culture dish, and with many smaller cells of the oligodendrocyte lineage on their surface. Dorsal root ganglia placed on these cultures grow axons readily, the overall extent of growth being unaffected by the presence or absence of oligodendrocytes, many of which express galactocerebroside and the inhibitory molecule janusin. A previous set of experiments had shown that growth of these axons is inhibited by oligodendrocytes by themselves. Scanning electron microscopy coupled with silver-intensified immunostaining reveals that the axons grow on the surface of the astrocytic layer, under-neath the oligodendrocytes, and are therefore in contact with both cell types as they grow. The presence of astrocytes therefore alters the results of axonal contact with oligodendrocytes.
Statistical evidence for a random commitment of pluripotent cephalic neural crest cells
Authors: Baroffio Anne , Blot Michel
Published: April 2021

The neural crest (NC) of vertebrate embryos yields cell types belonging to the neural, melanocytic and mesec-todermal lineages. To test the possibility that the precursors of these lineages segregate from pluripotent cells by a process involving stochastic determinants, we have analyzed with statistical methods the associations between six differentiated cell types in 201 clones obtained in vitro from migrating cephalic NC cells. Our analysis suggests that neuronal, adrenergic and Schwann cells are not randomly associated, whereas these neural cell types differentiate in the clones independently of both melanocytes and cartilage. These results raise the possibility that pluripotent NC progenitors give rise to the precursors of the major NC-derived lineages (neural, melanocytic and mesectodermal) by a process involving stochastic restrictions of their developmental potentialities.
Characterization of the human nestin gene reveals a close evolutionary relationship to neurofilaments
Authors: Dahlstrand Jonas , Zimmerman Lyle B. , McKay Ronald D.G. , Lendahl Urban
Published: April 2021

Multipotential stem cells in the neural tube give rise to the different neuronal cell types found in the brain. Abrupt changes in intermediate filament gene expression accompany this transition out of the precursor state: transcription of the intermediate filament nestin is replaced by that of the neurofilaments. In order to identify human neural precursor cells, and to learn more about the evolution of the intermediate filaments expressed in the central nervous system, we have isolated the human nestin gene. Despite considerable divergence between the human and rat nestin genes, in particular in the repetitive parts of the carboxyterminal region, the positions of the introns are perfectly conserved. Two of the three intron positions are also shared by the neurofilaments, but not by other classes of inter-mediate filaments. This implies that nestin and the neu-rofilaments had a common ancestor after branching off from the other classes of intermediate filaments, and that nestin separated from the neurofilament branch before the different neurofilament genes diverged. The characterization of human nestin also facilitates the identification of human multipotential neural precursor cells. This will be of importance for central nervous system (CNS) tumor diagnosis and transplant-based clinical approaches to human neurodegenerative diseases.
Further characterisation of the talin-binding site in the cytoskeletal protein vinculin
Authors: Gilmore A.P. , Jackson P. , Waites G.T. , Critchley D.R.
Published: April 2021

The cytoskeletal protein vinculin is a component of adherens-type junctions where it is one of a number of interacting proteins thought to link the cytoplasmic domain of adhesion receptors to F-actin. Vinculin has been shown to bind to at least three other cytoskeletal proteins, talin, paxillin and a-actinin. In this study, we further characterise the talin-binding domain in vin-culin using a series of chick vinculin polypeptides expressed as glutathione-S-transferase fusion proteins in Escherichia coli. Thus 125I-talin bound to a fusion protein spanning residues 1-398, but not to those spanning residues 399-881 or 881-1066 in an SDS-PAGE gelblot assay. We have previously characterised two chick vinculin cDNAs (2.89 kb cDNA and cVin5) which are identical in the region of overlap except that cVin5 lacks coding sequence for residues 167-207. Interestingly, a fusion protein spanning residues 1-398, but lacking residues 167-207, was unable to bind talin. However, further analysis showed that residues 167-207 are insufficient to support binding, and deletion of as few as 31 N-terminal residues abolished binding activity. The results of the gel-blot assay were essentially confirmed using purified fusion proteins adsorbed to glutathioneagarose beads. The smallest vinculin fusion protein able to bind talin contained residues 1-258. This fusion protein was as effective as whole vinculin in inhibiting the binding of 125I-vinculin to talin-coated microtitre wells. Interestingly, mutations which altered the charge characteristics of the highly conserved residues 178 and 181 abolished binding, whereas conservative substitutions were without effect. However, such mutations did not abolish the ability of mutant polypeptides spanning residues 1-398 to target to cell-matrix junctions in Cos cells. We have investigated the possible origin of the cDNA clone cVin5 by defining the structure of a 5 portion of the chicken vinculin gene, and by analysing vin-culin transcripts in a variety of adult tissues and embryonic fibroblasts using reverse transcriptase and polymerase chain reaction. Although residues 167-207 are encoded on a separate exon, we have been unable to identify a tissue where this exon is alternatively spliced.
The tight association of the tyrosine kinase substrate annexin ii with the submembranous cytoskeleton depends on intact p11- and Ca2+-binding sites
Authors: Thiel Carsten , Osborn Mary , Gerke Volker
Published: April 2021

Annexin II, a member of the annexin family of Ca2+- and lipid-binding proteins, is a major substrate of the pp60src kinase. It is unique within the annexin protein family, since it can form a tight heterotetrameric complex with the cellular protein ligand p11, a member of the S100 protein family. Within the cell, the annexin II2p112 complex is localized at the cytoplasmic surface of the plasma membrane in the submembranous cytoskeleton. This intracellular localization is thought to be the consequence of a typical annexin II property observed in vitro, its Ca2+-dependent binding to phospholipids and cytoskeletal elements (F-actin, non-ery-throid spectrin). We employed site-directed mutagenesis to create mutant annexin II molecules with defects either in the p11-binding site or in the Ca2+-binding sites present in repeats 2, 3 and 4. The mutated annexin II derivatives were expressed in HeLa and RMCD cells by transfection of the appropriate DNA constructs in order to analyze the importance of p11- and Ca2+-binding for the intracellular localization of annexin II. Immunofluorescence microscopy with a monoclonal antibody that specifically detected the transfected annexin II derivatives indicated that the Ca2+-dependent incorporation of annexin II into the submembranous network depended on its ability to form the annexin II/p11 complex and on the presence of intact Ca2+-binding sites. Neither monomeric annexin II lacking an intact p11-binding site, nor the annexin II mutant with defects in the Ca2+- binding sites in repeats 2, 3 and 4 were associated with the Triton X-100-resistant network of the submembra-nous cytoskeleton.
Altered glycosylation and cell surface expression of β1 integrin receptors during keratinocyte activation
Authors: Kim Lawrence T. , Ishihara Shuichi , Lee Chong-Chou , Akiyama Steven K. , Yamada Kenneth M. , Grinnell Frederick
Published: April 2021

We studied the mechanism by which cell adhesiveness becomes activated when keratinocytes are removed from skin and placed into cell culture. Our results suggest that activation involves altered β1 integrin subunit glycosylation accompanied by an increase in cell surface 01 integrin receptors. Activated keratinocytes contained two forms of the β1 integrin subunit, ∼93 kDa and ∼113 kDa. As shown by pulse-chase experiments, the smaller represented the cytoplasmic precursor of the larger, and only the 113 kDa mature form was detected in integrin receptors expressed at the cell surface. Preactivated keratinocytes contained 01 integrin subunits ranging from ∼97 to 110 kDa. These β1 subunits had been processed through the Golgi, based on resistance to endoglycosidase-H treatment, and were not converted to 113 kDa subunits during subsequent cell culture. Experiments with endoglycosidase-F showed that differences in the apparent sizes of β1 integrin subunits observed in pre-activated and activated keratinocytes could be attributed to differences in subunit glycosylation. Smaller β1 subunits found in pre-activated keratinocytes, like the precursor 01 subunits of activated cells, appeared to be less efficient in reaching the cell surface. Overall, a ∼10-fold increase in the level of cell surface integrin receptors occurred concomitant with the increased proportion of 113 kDa 01 subunits found in activated cells. Endoglycosidase-F experiments also indicated that there were changes in keratinocyte a subunits associated with 01. In related experiments, keratinocytes cultured in low Ca2+, serum-free MCDB medium for 4 days proliferated but their adhesiveness did not become activated. Therefore, keratinocyte proliferation and activation of adhesion are regulated separately. Finally, substantial activation of keratinocytes was observed when serum was added to cells cultured in MCDB with serum, indicating a role for serum factors in the activation process.
Integrin expression and differentiation in transformed human epidermal cells is regulated by fibroblasts
Authors: Kaur P. , Carter W. G.
Published: April 2021

Normal human foreskin keratinocytes (HFKs) and transformed HFKs (FEPE1L-8 cells) generated by the introduction of cloned human papillomavirus type 16 sequences were compared for the expression and function of a family of adhesion receptors termed integrins. Initially, cells were examined in conventional monolayer cultures. FEPE1L-8s expressed integrins α1β1, α2β1, α3β1, α5β1, α6β4 and β1 at comparable levels to HFKs. Further, these receptors were fully functional in mediating specific interactions with exogenously supplied ligands. However, FEPE1L-8s exhibited decreased synthesis of a number of extracellular matrix components, including laminin, fibronectin and epiligrin, compared to normal HFKs, which may be an alternate mechanism for regulating adhesion. Subsequently, organotypic cultures (OCs), which provide a suitable in vitro model system for the ordered stratification and differentiation of keratinocytes, were used to study the regulation of integrins and various epidermal markers in normal and transformed cells. OCs consisted of keratinocytes plated on a collagen gel containing primary human fibroblasts, grown at an air-medium interface. Unlike normal HFKs, the transformed FEPE1L-8 cells exhibited (a) disorganized stratification and limited differentiation capacity, (b) invasion into the collagen gel, and (c) unregulated expression of α3β1 and α2β1, and underexpression of α6β4 integrins. Ordered stratification and spatial regulation of integrin expression could be induced in the FEPE1L-8s by substituting Swiss 3T3 fibroblasts in the collagen gel. Further data indicate that the human fibroblasts induce the transformed HFKs to invade into the collagen gel. We conclude that stromal cells play an important role in regulating (a) the differential expression of integrins, (b) the normal stratification and differentiation, and (c) the invasive behaviour of epidermal cells.
The small gtp-binding protein rab6p is distributed from medial golgi to the Trans-golgi network as determined by a confocal microscopic approach
Authors: Antony Claude , Cibert Christian , Géraud Gérard , Maria Angelica Santa , Maro Bernard , Mayau Véronique , Goud Bruno
Published: April 2021

A key role in the regulation of membrane traffic is played by the rab proteins, members of a family of ras-related small GTP-binding proteins. This family comprises at least 25 identified members, the intracellular localization of only a few of which has been investigated. rab6p has been shown to be distributed along the exo-cytic pathway in association with the medial and trans regions of the Golgi apparatus. A confocal laser scanning microscopic (CLSM) approach coupled with image analysis was used to compare the localization of rab6p with selected reference Golgi markers by double immunofluorescence on culture cell lines. CLSM analysis shows that, under a set of well-defined conditions, one can investigate the possible colocalization of known markers of Golgi compartments and orientate a couple of labeled Golgi antigens with regard to the polarity of the Golgi apparatus. Thus, having validated the CLSM analysis, the localization of rab6p was studied and compared with some of these markers and the VSV-G protein in VSV (vesicular stomatitis virus)-infected cells blocked at 20°C. rab6p is shown to be associated in all the cell lines used with the last cisternae of the Golgi apparatus and particularly with the trans-Golgi network (TGN), the site of protein sorting at the exit of the Golgi apparatus. These results were supported by an electron microscopic study using double-immunolabeled cryosections: rab6p was found in some flat cisternae of the Golgi stack and colocalized with the VSV-G protein in the TGN. Our results show that the small GTP-binding protein rab6p is distributed from medial Golgi to TGN along the exocytic pathway.
Fluorescent phospholipids preferentially accumulate in sub-tegumental cells of schistosomula of Schistoxssoma mansoni
Authors: Furlong Stephen T. , Thibault Kathleen S. , Rogers Rick A.
Published: April 2021

Schistosomes do not make sterols or fatty acids de novo and thus require host lipids for survival. The acquisition of host lipids may also be an important factor in the schistosome’s defense from host immunity; however, little is known about the regulation of this process. Here we have examined binding of radiolabeled and fluorescently labeled liposomes to schistosomula, and followed incorporation of fluorescent phospholipids into the worm by both morphological and biochemical methods. Saturable binding of radiolabeled phosphatidylcholine containing liposomes was observed and epifluorescence microscopy showed binding of C6-NBD-phosphatidyl-choline (C6-NBD-PC), C12-NBD-phosphatidylcholine (C12-NBD-PC) and C6-NBD-phosphatidyl-ethanol-amine (C6-NBD-PE) containing liposomes on the surface of the parasite. Following back-exchange with unlabeled liposomes, NBD-PC and NBD-PE were observed to be preferentially incorporated into specific cell types within the worm. Furthermore, cells which had accumulated the fluorescent lipid formed an interconnecting cellular network immediately below the tegument, identified as cytons. By contrast, fluorescein-PE was found only on the surface of the parasite and in the gut but not in the cytons. Biochemical analysis demonstrated that >90% of the C6-NBD-PC and C12-NBD-PC remained as the intact molecule after a one hour incubation with the parasite, but that greater than 70% of the NBD-PE was converted to other lipids. These studies demonstrate that incorporation of phospholipid analogs into schistosomula can be followed morphologically and biochemically. As there was little localization of NBD-PE or NBD-PC in the gut, these analogs must be assimilated by crossing the tegument. Furthermore, specificity in the worm’s uptake processes are evident.
Preferential mitochondrial and plastid dna synthesis before multiple cell divisions in Nicotiana tabacum
Authors: Suzuki Takeshi , Kawano Shigeyuki , Sakai Atsushi , Fujie Makoto , Kuroiwa Haruko , Nakamura Hiromu , Kuroiwa Tsuneyoshi
Published: April 2021

Organelle DNA synthesis in root meristem and cultured cell line BY-2, both derived from Nicotiana tabacum cv. Bright Yellow 2, was examined by immunofluorescence microscopy of Technovit sections with antibody against 5-bromodeoxyuridine (BrdU) and co-fluorescent staining with 4′,6-diamidino-2-phenylindole (DAPI) and quantitative Southern hybridization. In the root meristem, the mitochondrial DNAs (mtDNAs) were synthesized in a specific region near to the quiescent center, where a low frequency of DNA synthesis of cell nuclei was observed. The mitochondrial nuclei (nucleoids) changed morphologically from long ellipsoids with a high frequency of DNA synthesis, in the region just above the quiescent center, to granules with a low frequency of DNA synthesis, as cell distance from the quiescent center increased. Similar patterns were observed in the cultured tobacco cell line (BY-2), in which large amounts of preferential synthesis of DNA of both mitochondria and plastids occurred prior to cell nuclear DNA synthesis just after stationary phase cells were transferred to fresh medium. Granular mitochondria which vigorously synthesized mtDNA were observed in both lag phase and logarithmic growth phase cells. However, long, ellipsoidal mitochondria which showed a low frequency of mtDNA synthesis were observed in stationary phase cells. Morphological changes of plastids were more conspicuous than those of mitochondria. After the medium was renewed, spherical plastids became extremely elongated and string-like, for 24 h, but were divided into small pieces after the third day. Vigorous synthesis of plastid DNA (ptDNA) occurred during this period of plastids elongation.
Expression of wild-type and nuclear localization-deficient human lamin a in chick myogenic cells
Authors: Lourim David , Lin Jim Jung-Ching
Published: April 2021

Previous analysis of chick embryonic muscle (CEM) differentiation in vivo and in ovo demonstrated that lamin A accumulation to steady-state levels preceded the accumulation of muscle-specific proteins. These observations have suggested the appearance of A-type lamins may be important for differentiation. To test this hypothesis, we have temporally and quantitatively altered the expression of A-type lamins in CEM cells by transient transfection of wild-type (wt; pHLA) or nuclear localization-deficient (NLd; pHLA-del) human lamin A expression plasmids. Transfected CEM cells synthesized the wt and NLd human lamin As to high levels, both of which were resistant to high-salt extraction. The wt human lamin A localized to the nucleus, whereas the NLd protein showed cytoplasmic staining patterns, as well as time-dependent nuclear localization. The presence of endogenous chicken lamins A and B2 in NLd human lamin A cytoplasmic structures suggested the interspecies lamin copolymerization. Thus, this approach may provide a possible method for analysis of lamin-lamin or lamin-lamina component interactions in vivo. With regard to muscle differentiation, CEM cells transfected with either pHLA or pHLA-del demonstrated moderate and transient increased levels of the muscle-specific myosin heavy chain and creatine kinase activity. These increases appeared temporally and quantitatively to reflect the transient accumulation of the human lamin As. In contrast, β-tubulin and skeletal tropomyosin protein accumulations appeared unaffected. On the basis of these results, we suggest that nuclear lamina content and structure may play a limited, permissive and indirect role in the temporally regulated expression of the myogenic phenotype.
Flagellar, cellular and organismal polarity in Volvox carteri
Authors: Hoops Harold J.
Published: April 2021

It has previously been shown that the flagellar apparatus of the mature Volvox carteri somatic cell lacks the 180° rotational symmetry typical of most unicellular green algae. This asymmetry has been postulated to be the result of rotation of each half of the flagellar apparatus. Here it is shown that V. carteri axonemes contain polarity markers that are similar to those found in Chlamydomonas, except that in V. carteri the number one doublets do not face each other as they do in Chlamydomonas but are oriented in parallel and at approximately right angles to the line that connects the flagella. Thus, the rotational orientations of the axonemes are consistent with the postulate that the flagella of V. carteri have rotated in opposite directions, as was predicted earlier from the positions of the basal fibers and microtubular rootlets. Moreover, high-speed cinephotomicrographic analysis shows that the V. carteri flagellar effective strokes are also oriented in approximately the same direction, and in parallel planes. These results suggest that the direction of the effective stroke in both Chlamydomonas and Volvox is fixed, and that rotation of the axoneme is the cause of the differences in flagellar motility observed between Chlamydomonas and Volvox. These differences are probably essential for effective organismal motility. Cellular polarity of V. carteri can be related to that of Chlamy domonas after taking into account the developmental reorientation of flagellar apparatus components. This reorientation also results in the movement of the eyespot from a position nearer one of the flagellar bases to a position approximately equidistant between them. By analogy to Chlamydomonas, the anti side of the V. car teri somatic cell faces the spheroid anterior, the syn side faces the spheroid posterior. The cis side of the cell is to the cell’s left (the right to an outside observer), although it cannot be described solely on the basis of eyespot position as it can in Chlamydomonas, while the trans side is to the cell’s right. It follows that if the direction of the effective flagellar stroke is specified by structural features, then effective organismal motility in V. carteri, will be accomplished only if the cells are held in the proper orientation with respect to one another. The simplest arrangement that will yield both progression and rotation in ovoid or spherical colonies composed of biflagellate isokont cells is one in which the cells are arranged with rotational symmetry about the anteriorposterior axis of the spheroid. Analysis of the polarity of somatic cells from throughout the spheroid shows that it is constructed with just such symmetry. This symmetry probably originates with the very first divisions.
A survey of interactions made by the giant protein titin
Authors: Soteriou A. , Gamage M. , Trinick J.
Published: April 2021

A simple solid-phase binding assay was used to screen for interactions that the giant myofibrillar protein titin makes with other sarcomeric proteins. The titin used in the tests was purified by a modified procedure that results in isolation of ∼20 mg relatively undegraded protein in <24 h. In addition to the ∼3 MDa polypeptide, bands at ∼160 kDa and ∼100 kDa were also consistently seen on gels. Binding of titin to myosin, C-protein, X-protein and AMP-deaminase was observed. The interaction with myosin appears to be with the light meromyosin part of the molecule.
Microinjection of a monoclonal antibody against SPN antigen, now identified by peptide sequences as the NuMA protein, induces micronuclei in PtK2 cells
Authors: Kallajoki Markku , Harborth Jens , Weber Klaus , Osborn Mary
Published: April 2021

Several high molecular mass proteins which relocate from the interphase nucleus to the spindle poles during mitosis have been defined by antibodies. Microinjection experiments have shown that at least the antigen defined by SPN antibody plays a functional role during mitosis. Recently the cDNA sequence for human NuMA antigen was established and epitopes for antibodies to centrophilin, and to 1F1 and 1H1 antigens were found to be included in the NuMA protein. Here we show that immunoprecipitated SPN antigen reacts with an autoimmune human NuMA serum. In addition three peptides derived from immunoprecipitated human SPN by cyanogen bromide cleavage and covering more than fifty amino acids show a perfect fit with the sequence predicted for NuMA protein. Thus SPN antigen and NuMA are the same protein. Injection of SPN-3 antibody into interphase or mitotic PtK2 cells results in cells with micronuclei. For cells injected in prophase, prometaphase or metaphase 90%, 78% and 77% display defective cytokinesis or yield daughter cells with micronuclei. In contrast only 16% of cells injected in anaphase are abnormal. Thus SPN/NuMA antigen may be required during early, but not during later, stages of mitosis. Surprising parallels are seen between the effects of microinjecting SPN-3 antibody and treatment with colcemid and taxol of PtK2 and HeLa cells. Our results identify an important role during mitosis for the SPN/NuMA antigen.
Lytic granules from cytotoxic T cells exhibit kinesin-dependent motility on microtubules in vitro
Authors: Burkhardt J. K. , McIIvain J. M. , Sheetz M. P. , Argon Y.
Published: April 2021

One major mechanism of cell-mediated cytolysis is the polarized secretion of lytic granules, a process which is highly dependent on microtubules. We isolated lytic granules from murine cytotoxic T cells and tested their ability to bind to and move along microtubules in vitro. In the presence of a motor-containing supernatant, the granules bound to the microtubules and moved along them at an average maximal rate of 1 m/second. Virtually every granule could bind to microtubules, and about half translocated within a few seconds of binding. Motility required exogenous cytosolic motors, hydrolyzable nucleotides, and an intact granule membrane. Although the motor preparation used to support granule movement contains both plus- and minus-enddirected motor proteins, granule movement was strongly biased toward microtubule plus-ends. Inactivation of cytoplasmic dynein had little effect on granule binding and movement, but immuno-depletion of kinesin from the motor preparation inhibited granule binding by 50%. These results indicate that most granule movement in this assay is mediated by kinesin. The speed and direction of granule movement in vitro are sufficient to account for the release of lytic granules in the intact T cell. This model system should be valuable for studying the interactions of secretory granules with microtubules, and for identifying the regulatory factors involved.
Haemopoietic stem cell development to neutrophils is associated with subcellular redistribution and differential expression of protein kinase C subspecies
Authors: Shearman M. S. , Heyworth C. M. , Dexter T. M. , Haefner B. , Owen P. J. , Whetton A. D.
Published: April 2021

Multipotential FDCP-Mix A4 (A4) cells can be induced either to self-renew or to differentiate and develop into mature neutrophils in liquid culture, depending on the haemopoietic growth factors with which they are cultured. When cultured in low concentrations of interleukin 3 (IL-3, 1 unit/ml)) plus Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Granulocyte-CSF (G-CSF), A4 cells proliferate with accompanying development to form cells which resemble mature, postmitotic neutrophils. The presence of high concentrations of IL-3 (100 units/ml) blocks the development of A4 cells even in the presence of GM-CSF plus G-CSF. A4 cell development to neutrophils is accompanied by major changes in the expression of protein kinase C (PKC) subspecies in these cells. The predominant subspecies present in multipotent A4 cells, as judged by direct chromatographic analysis, was the type III enzyme (α) subspecies, whereas in mature A4 cell neutrophils, the type II (βI + βII) enzymes were predominant. Phorbol esters added to immature A4 cells resulted in a proliferative response, but when added to postmitotic A4 cells resembling neutrophils they elicited a large increase in reactive oxygen intermediate production. This suggests that the type III (α) subspecies may mediate proliferative responses in stem cells, whilst the type II (βI + βII) enzymes are more important for the mature cell functions of postmitotic neutrophils. In cultures containing IL-3 (100 units/ml) both the type III, and also the type II subspecies were predominantly membrane-associated for prolonged periods (>24 hours). The addition of IL-3 (100 units/ml) to FDCP-Mix A4 cells starved of haemopoietic growth factors led to the rapid translocation of protein kinase C from the cytosol to the membrane; no such effect was observed with GM-CSF or 1 unit/ml IL-3. Under conditions where differentiation and development were induced (1 unit/ml of IL-3 plus GM-CSF and G-CSF), there was a redistribution of all PKC subspecies to the cytosol from the membrane. Thus, IL-3 preserves the multipotential nature of A4 cells and translocates PKC to the membrane. As GM-CSF cannot stimulate the translocation of protein kinase C, the differential biochemical and developmental effects of these growth factors on multipotent cells may in part be mediated by the activation of protein kinase C.
Chemotactic antagonists of cAMP inhibit Dictyostelium phospholipase C
Authors: Bominaar Anthony A. , Haastert Peter J. M. Van
Published: April 2021

In Dictyostelium discoideum extracellular cAMP induces chemotaxis via a transmembrane signal transduction cascade consisting of surface cAMP receptors, G-proteins and effector enzymes including adenylyl cyclase, guanylyl cyclase and phospholipase C. Previously it was demonstrated that some cAMP derivatives such as 3′-deoxy-3′ -aminoadenosine 3′ :5′ -monophosphate (3 NH-cAMP) bind to the receptor and induce normal activation of adenylyl cyclase and guanylyl cyclase. However these analogues do not induce chemotaxis, probably because the signal is transduced in an inappropriate manner.

We have now studied the regulation of phospholipase C by cAMP and these chemotactic antagonists. cAMP induced the two-fold activation of phospholipase C leading to a transient increase of Ins(1,4,5)P3 levels. In contrast, the analogues induced a rapid decrease of intracellular Ins(1,4,5)P3 levels, due to the inhibition of phospholipase C activity. In a transformed cell-line lacking the G-protein that mediates phospholipase C inhibition, 3′ NH-cAMP did not decrease phospholipase C activity and was no longer an antagonist of chemotaxis. These results suggest that inhibition of phospholipase C leads to aberrant chemotaxis.
cDNA-derived molecular characteristics and antibodies to a new centrosome-associated and G2/m phase-prevalent protein
Authors: Rothbarth Karsten , Petzelt Christian , Lu Xiang , Todorov Ivan T. , Joswig Gaby , Pepperkok Rainer , Ansorge Wilhelm , Werner Dieter
Published: April 2021

Differential screening of a murine RNA-based cDNA library with cell cycle phase-specific transcripts released a cDNA clone (CCD41) to a mRNA (1.349 kb) which, according to the mode of its detection, increases as expected during the cell cycle. The molecular characteristics of the protein (27 103Mr) encoded by this mRNA were deduced from the cDNA sequence and antibodies were prepared against the recombinant protein. Immunofluorescence studies performed with PtK2 cells revealed that the amount of the antigen specified by the CCD41 sequence increases during the cell cycle out of proportion with the DNA content. In G1 phase cells, the antigen is exclusively located at the site of the centrosome. During cell cycle progression the antigen becomes also detectable in perinuclear vesicles that increase in number and size, reaching a maximum in G2 phase cells. The centrosomal location of the CCD41 antigen was investigated in relation to another centrosomal antigen, centrosomin A. Since the latter antigen is detected by a monoclonal antibody reacting specifically and permanently with the centrosomes in PtK2 cells throughout the cell cycle it was possible to investigate the relative positions of the two proteins at the site of the centrosome and to add new information about the general architecture of the organelle and its changes during the cell cycle. While the centrosomin A antibody detects the pronounced cell cycle stage-dependent shape changes of the centrosome, the CCD41-encoded protein appears to be localized as a compact structure inside the centrosome. Its epitopes are exposed throughout the cell cycle except during a brief period immediately after the formation of the daughter centrosome.
A study of the structure, function and distribution of β5 integrins using novel anti-β5 monoclonal antibodies
Authors: Pasqualini Renata , Bodorova Jana , Ye Song , Hemler Martin E.
Published: April 2021

Here, we have utilized six new anti-human β5 mono-clonal antibodies to perform a detailed investigation of the structure, function and distribution of β5 integrins. Monoclonal anti-β5 specificity was confirmed by reac-tivity with β5-transfected CHO cells, by direct binding to the β5 subunit (immunoblotting), and by immunode-pletion experiments using polyclonal anti-β5 serum. The β5 subunit was predominantly associated with the av subunit, although on some cell lines, the level of β5 exceeded that of αv for unknown reasons. Cell adhesion studies showed that the adhesive function of β5 could be stimulated, inhibited or unaltered by different anti-β5 monoclonal antibodies. The β5 subunit was involved in adhesion to both vitronectin and fibronectin and, at least for K562 cells, fibronectin appeared to be the preferred ligand. Flow-cytometry studies showed that the β5 subunit was expressed at moderate to high levels on all adherent cell lines examined, was absent from all lym-phoid cell lines, and was only weakly expressed on myeloid cell lines. Staining of thymic sections showed the distribution of β5 on blood vessels, Hassal’s corpus-cles, cortical and medullary stromal cells, and basement membranes. Skin sections showed β5 on the basal layer of the epidermis and on some dermal blood vessel walls, and kidney sections showed staining of glomerular regions, juxta glomerular apparatus, proximal convo-luted tubules and collecting tubules, and at least one anti-β5 antibody also stained epithelial cells of proximal tubules.
Inhibition of transcription blocks cell cycle progression of NIH3T3 fibroblasts specifically in G1
Authors: Adolph Sabine , Brüsselbach Sabine , Müller Rolf
Published: April 2021

We have analysed the role of RNA polymerase II-depen-dent transcription in cell cycle progression. Time-lapse video recording and cytogenetic analysis were used to determine the sensitivity of NIH3T3 cells to the RNA polymerase II inhibitor -amanitin at different stages of the cell cycle. Our results show that -amanitin blocks cells specifically in G1, irrespective of the concentration within the range of 3 to 30 g/ml. This indicates that transcription in G1 is required to overcome a restriction point located in this phase of the cell cycle. In agree-ment with this conclusion is the requirement for an uninhibited protein synthesis during G1 progression. In addition, the insensitivity of S-phase cells to RNA poly-merase II inhibition suggests that the transcription of genes thought to be normally induced during S/G2 is not required for the completion of an ongoing cell cycle. S/G2 progression was however clearly dependent on protein synthesis. This suggests that cells exposed to -amanitin are able to complete their cell cycle because sufficiently high levels of mRNA are present in S/G2 due to basal level transcription, or are left from preceding cell cycles. It is therefore unlikely that transcriptional regulation in S or G2 plays a crucial role in the control of cell cycle progression in NIH3T3 cells.