bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stimulation of calcium influx by platelet activating factor in Dictyostelium
Authors: Schaloske Ralph , Sordano Concetta , Bozzaro Salvatore , Malchow Dieter 
Published: April 2021

ABSTRACT
Platelet activating factor (PAF) induces Ca2+ influx in Dictyostelium discoideum. In this investigation we used this activity to analyze the mechanism of PAF action. We found that PAF activity was confined to the period of spike-shaped oscillations and suggest that the role of PAF is to augment cAMP relay. PAF seems to act only a few times during this time period of two hours, since Ca2+ entry adapted to a subsequent stimulus for about 30 minutes. PAF showed a reduced response in the G protein β− strain LW14 and was unable to induce Ca2+ influx in the Gα2− strains HC85 and JM1. The latter expresses the cAMP receptors cAR1 constitutively, and exhibits cAMP-induced Ca2+ influx, albeit at a reduced level. In order to decide whether the inability of PAF to elicit a Ca2+ response in JM1 cells was due to the lack of differentiation and/or the lack of Gα2, we inhibited the IP3-dependent pathway with compound U73122 and found that Ca2+ entry was blocked, whereas a closely related inactive compound, U73343, did not alter the response. In agreement with this, NBD-Cl, an inhibitor of Ca2+ uptake into the IP3-sensitive store in Dictyostelium, also abolished PAF activity. The latter was not inhibited by the plasma membrane antagonists BN-52021 or WEB 2170. Therefore PAF seems to operate intracellularly via the IP3-signalling pathway at or upstream of the IP3-sensitive store.
Regulated secretion of multi-lamellar vesicles leads to formation of a tubulo-vesicular network in host-cell vacuoles occupied by Toxoplasma gondii
Authors: Sibley L. David , Niesman Ingrid R. , Parmley Steve F. , Cesbron-Delauw Marie-France 
Published: April 2021

ABSTRACT
Toxoplasma gondii is an obligate intracellular parasite that actively invades virtually all types of nucleated cells, surviving within a specialized vacuole called the para-sitophorous vacuole. Shortly after invasion, the parasite modifies this vacuole by secreting a variety of proteins from electron-dense storage granules. Additionally, the parasite forms a network of membranous tubules within the lumen of the vacuole and connecting with the vacuolar membrane. We have used immunolabeling and cell fractionation to examine the secretion of two dense granule proteins, GRA1 and GRA2, which are involved in formation of the intravacuolar network. Following host-cell invasion, GRA1 was secreted into the lumen of the vacuole as a soluble protein that subsequently became peripherally associated with the network. In addition to being secreted as a soluble protein from dense granules, GRA2 was secreted within multi-lamellar vesicles released from a specialized posterior invagination of the parasite. The multi-lamellar vesicles assemble to form the intravacuolar network, which contains an integral membrane form of GRA2. These findings indicate that Toxoplasma has a highly developed regulated exocytosis pathway that modifies the para-sitophorous vacuole by secretion of soluble proteins and by a novel process of membrane secretion.
The expression of carbohydrate blood group antigens correlates with heat resistance
Authors: Ménoret Antoine , Otry Caroline , Labarrière Nathalie , Breimer Michael E. , Piller Friedrich , Meflah Khaled , Pendu Jacques Le 
Published: April 2021

ABSTRACT
Recent data indicate that cells may resist heat shock via more than one route: heat shock protein synthesis and other still ill-defined mechanisms. We investigated this phenomenon using four types of cells derived from a single rat colon carcinoma: clones REGb and PROb; PRO A+, a glycosylation variant of PROb selected for its high expression of blood group A antigen; and Ph8, a thermoresistant variant of PROb selected by repeated sublethal heat treatments. Basal heat resistance was clearly associated with the level of cell surface expression of blood group H and A antigens. Biosynthesis of these carbohydrate structures requires two glycosyltransferases, H and A enzymes, whose activities are also correlated with basal heat resistance. In addition, heat sensitive REGb cells were rendered more resistant by transfection with the gene encoding for H enzyme, allowing expression of H antigen. Thus, these terminal glycosylations could play a role as cellular protectors against heat treatment. Blood group carbohydrate antigens were mainly located on O-linked carbohydrate chains of a major glycoprotein of 200 kDa and to a lesser extent on N-linked chains. Only trace amounts were present as glycolipids.
The inductive effect of a human DNA sequence (HALF1) on the differentiation of a variant rat hepatoma cell (C2) is restricted to episomal forms of the molecule
Authors: Ng Kim Heng , Maigné Joèlle , Deschatrette Jean 
Published: April 2021

ABSTRACT
HALF1, a 4.3 kb human DNA sequence, was originally identified as a double-stranded, closed-circular DNA molecule in revertants from a dedifferentiated rat hepatoma cell (C2) transfected with human liver DNA. Here we report its specific properties in inducing the transition to the hepatic phenotype. (i) In vitro recircularized HALF1 induces reversion after a minimum time lag of 7 days post-transfection. (ii) After induction, the presence of HALF1 is not required for maintaining the induced hepatic state. (iii) HALF1 is detected as a sequence integrated in high molecular mass DNA of human liver. (iv) HALF1 monomer or dimer plasmid constructs do not induce reversion when integrated into the genome of transfectants. (V)Short ubiquitous RNA transcripts (∼400 bases) are detected with specific HALF1 probes.
These results indicate that the reversion process is linked to the presence of HALF1 extrachromosomal molecules.
Maturation of early endosomes and vesicular traffic to lysosomes in relation to membrane recycling
Authors: Thilo Lutz , Stroud Evelyn , Haylett Thomas 
Published: April 2021

ABSTRACT
The controversy whether endocytic processing occurs by organellar maturation or by vesicular traffic has not been resolved. It is also not clear whether maturation continues to the stage of lysosomes, to what extent it involves a decrease in organellar fusogenicity, and how it relates to membrane recycling. Maturation and vesicular traffic imply distinct kinetics for the intermingling of endocytic markers after sequential endocytic uptake. We have studied the kinetics of intermingling of fluid-phase markers (fluorescein-labelled dextran and horseradish peroxidase) and cell surface-derived membrane (labelled by galactosylation) in organelles at early and late stages of the endocytic pathway in macrophage-like P388D1 cells. Intermingling declined by sigmoid kinetics, indicating that endosomes matured within about 3 minutes to become non-fusogenic towards early endosomes. During maturation about 60% of internalized membrane was recycled with TG ≈2 minutes. Whereas matured endosomes were non-fusogenic towards early endosomes and towards each other, a second phase of intermingling was observed upon delivery to lysosomes. This intermingling occurred by a first-order process (TG ≈4 minutes), concurrent with recycling of the remaining 40% of internalized membrane marker. These kinetic observations suggest a model for endocytic processing which reconciles maturation of early endosomes with the known function of carrier vesicles: Endocytic carrier vesicles do not bud off from permanent early endosomes as proposed for vesicular traffic, but are derived, together with recycling vesicles, from the maturation of early endosomes which are consumed by this process; these carrier vesicles subsequently mediate delivery to lysosomes by vesicular traffic during which the remaining surface-derived membrane is recycled.
Efficient reactivation of Xenopus erythrocyte nuclei in Xenopus egg extracts
Authors: Wangh Lawrence J. , DeGrace Diane , Sanchez J. Aquiles , Gold Alex , Yeghiazarians Yerem , Wiedemann Karen , Daniels Susan 
Published: April 2021

ABSTRACT
Rapid genome replication is one of the hallmarks of the frog embryonic cell cycle. We report here that complete reactivation of quiescent somatic cell nuclei in Xenopus egg extracts depends on prior restructuring of the nuclear substrate and prior preparation of cytoplasmic extract with the highest capacity to initiate and sustain DNA synthesis. Nuclei from mature erythrocytes swell, replicate their DNA efficiently, and enter mitosis in frozen/thawed extracts prepared from activated Xenopus eggs, provided the nuclei are first treated with trypsin, heparin, and an extract prepared from unactivated, meiotically arrested, eggs. Optimal replicating extracts are prepared from large batches of unfertilized eggs that are synchronously activated into the cell cycle for 28 minutes (at 20°C). Because the Xenopus cell cycle progresses so rapidly, extracts prepared just a few minutes before or after this time have substantially lower DNA synthetic capacities. At the optimal time and temperature, eggs have just reached the G1/S boundary of the first cell cycle. This fact was revealed by injecting and replicating an SV40 plasmid in intact unfertilized eggs as described previously. We estimate that under optimal conditions approximately 6.14×109 base pairs of DNA/per nucleus are synthesized in 30-40 minutes, a rate that rivals that observed in the zygotic nucleus.
The findings reported here are one step in our long term effort to develop a new in vitro/in vivo approach to nuclear transplantation. Nuclear transplantation in amphibian embryos has been used to establish that the genomes of many types of differentiated somatic cells are pluripotent. But very few such nuclei have ever developed into advanced tadpoles or adult frogs, probably because somatic nuclei injected directly into activated eggs fail to reactivate quickly enough to avoid being damaged during first mitosis. We have already shown that unfertilized eggs can be injected prior to activation of the first cell cycle. Future experiments will reveal whether in vitro reactivated somatic cell nuclei transplanted into such eggs reliably reach advanced stages of development.
Co-localization of hydrolytic enzymes with widely disparate ph optima: Implications for the regulation of lysosomal ph
Authors: Butor Cécile , Griffiths Gareth , Aronson Nathan N. , Varki Ajit 
Published: April 2021

ABSTRACT
Lysosomes are traditionally defined by their acidic interior, their content of degradative ‘acid hydrolases’, and the presence of distinctive membrane proteins. Terminal degradation of the N-linked oligosaccharides of glycoproteins takes place in lysosomes, and involves several hydrolases, many of which are known to have acidic pH optima. However, a sialic acid-specific 9-O-acetyl-esterase and a glycosyl-N-asparaginase, which degrade the outer- and inner-most linkages of N-linked oligosaccharides, respectively, both have pH optima in the neutral to alkaline range. By immunoelectron microscopy, these enzymes co-localize in lysosomes with several conventional acid hydrolases and with lysosomal membrane glycoproteins. Factors modifying the pH/activity profiles of these enzymes could not be found in lysosomal extracts. Thus, the function of the enzymes with neutral pH optima must depend either upon their minimal residual activity at acidic pH, or upon the possibility that lysosomes are not always strongly acidic. Indeed, when lysosomes are marked in living cells by uptake of fluorescently labeled mannose 6-phosphorylated proteins, the labeled organelles do not all rapidly accumulate Acridine Orange, a vital stain that is specific for acidic compartments. One plausible explanation is that lysosomal pH fluctuates, allowing hydrolytic enzymes with a wide range of pH optima to efficiently degrade macromolecules.
Actin-dependent light-induced translocation of mitochondria and er cisternae in the photoreceptor cells of the locust Schistocerca gregaria
Authors: Stürmer Karoline , Baumann Otto , Walz Bernd 
Published: April 2021

ABSTRACT
Light-dependent changes in the positioning of organelles in photoreceptor cells of arthropods are a well-known phenomenon. In this study, we examine the role of the cytoskeleton in these light-dependent antagonistic movements. In dark-adapted photoreceptor cells of the locust Schistocerca gregaria, prominent sacs of smooth endoplasmic reticulum (ER) oppose the bases of the photoreceptive microvilli. Light stimulation causes a translocation of the ER elements towards the main cell body, and an aggregation of mitochondria adjacent to the microvilli. Immunofluorescence studies and electron-microscopic examination of chemically fixed or high-pressure-frozen, freeze-substituted specimens demonstrate a lack of microtubules in the submicrovillar region. However, numerous filament bundles are aligned in close association with mitochondria and ER elements, along the track of their movement. Fluorescent phallotoxins and monoclonal antiactin antibodies label filament bundles in the submicrovillar region, indicating that they are composed of F-actin. Finally, depolymerization of the submicrovillar actin filaments by incubation with cytochalasin B results in a blockade of the movement of mitochondria and ER cisternae towards the rhabdom. These results suggest that the light-dependent translocation of both ER cisternae and mitochondria occurs along actin filaments.
Different endocytic compartments are involved in the tight association of class ii molecules with processed hen egg lysozyme and ribonuclease a in b cells
Authors: Escola Jean-Michel , Grivel Jean-Charles , Chavrier Philippe , Gorvel Jean-Pierre 
Published: April 2021

ABSTRACT
The processing of exogenous antigens and the association of peptides with class II molecules both occur within the endocytic pathway. 2A4 B lymphoma cells of the H-2k haplotype were grown in the presence or the absence of two different exogenous antigens (hen egg lysozyme and ribo-nuclease A) internalized by fluid-phase endocytosis. Using subcellular fractionation techniques, we demonstrate that, in the presence of hen egg lysozyme, newly synthesized SDS-stable class II molecules are detected in a dense endocytic compartment which does not have the characteristics of neither early and late endosomes nor lysosomes. In contrast, no SDS-stable class II molecules are observed between ribonuclease A and newly synthesized class II molecules. Interestingly, when class II molecules are analyzed at steady state, SDS-stable class II molecules induced by ribonuclease A are found in a compartment cosedimenting with late endosomes. These results suggest that the tight associations between ribonuclease A or hen egg lysozyme with class II molecules occur in distinct endocytic compartments and that these associations may depend on the sensitivity of antigens to proteolysis.
Subcellular localisation of human wee1 kinase is regulated during the cell cycle
Authors: Baldin Véronique , Ducommun Bernard 
Published: April 2021

ABSTRACT
Wee1 kinase-dependent phosphorylation of cdc2 maintains the cdc2/cyclin B complex in an inert form until it is activated by the cdc25 tyrosine phosphatase at the end of G2. As described for cdc25, cell cycle-linked changes in the intracellular localisation of wee1 may constitute an important aspect of the temporal regulation of cdc2 activity. Here we report that the subcellular distribution of human wee1 changes during the cell cycle in HeLa and IMR90 cells. During interphase, wee1 is found almost exclusively in the nucleus. When the cell enters mitosis, wee1 is relocalised into the cytoplasm. During cell division, wee1 becomes restricted to the mitotic equator and by the end of mitosis it is found exclusively in association with midbody bridges, a phenomenon that is dependent on microtubule assembly. The relocalisations of wee1 and its association with subcellular structures may play key regulatory roles at different stages of the cell cycle and during mitosis.
Involvement of M-cadherin in terminal differentiation of skeletal muscle cells
Authors: Zeschnigk Michael , Kozian Detlef , Kuch Christine , Schmoll Marion , Starzinski-Powitz Anna 
Published: April 2021

ABSTRACT
Cadherins are a gene family encoding calcium-dependent cell adhesion proteins which are thought to act in the establishment and maintenance of tissue organization. M-cadherin, one member of the family, has been found in myogenic cells of somitic origin during embryogenesis and in the adult. These findings have suggested that M-cadherin is involved in the regulation of morphogenesis of skeletal muscle cells. Therefore, we investigated the function of M-cadherin in the fusion of myoblasts into myotubes (terminal differentiation) in cell culture. Furthermore, we tested whether M-cadherin might influence (a) the expression of troponin T, a typical marker of biochemical differentiation of skeletal muscle cells, and (b) withdrawal of myoblasts from the cell cycle (called terminal commitment). The studies were performed by using antagonistic peptides which correspond to sequences of the putative M-cadherin binding domain. Analogous peptides of N-cadherin have previously been shown to interfere functionally with the N-cadherin-mediated cell adhesion. In the presence of antagonistic M-cadherin peptides, the fusion of myoblasts into myotubes was inhibited. Analysis of troponin T revealed that it was downregulated at the protein level although its mRNA was still detectable. In addition, withdrawal from the cell cycle typical for terminal commitment of muscle cells was not complete in fusion-blocked myogenic cells. Finally, expression of M-cadherin antisense RNA reducing the expression of the endogenous M-cadherin protein interfered with the fusion process of myoblasts. Our data imply that M-cadherin-mediated myoblast interaction plays an important role in terminal differentiation of skeletal muscle cells.
Characterization of a 5.4 kb cDNA fragment from the Z-line region of rabbit cardiac titin reveals phosphorylation sites for proline-directed kinases
Authors: Sebestyén Magdolna G. , Wolff Jon A. , Greaser Marion L. 
Published: April 2021

ABSTRACT
Titin is an approximately 3 MDa protein that spans from the M- to the Z-line in the sarcomeres of vertebrate striated muscle. The protein is presumably encoded by unusually large mRNAs of 70-80 kb. Although titin has been studied by several laboratories, barely more than half of the cDNA sequence (≈45 kb) has been published, most of it obtained from the A-band and M-line region (corresponding to the C-terminal half of the molecule). A special cDNA library was constructed using size selected total RNA from adult rabbit cardiac muscle in order to obtain sequence data from titin’s unknown N-terminal region. A monoclonal antibody (T12), which binds to an epitope close to the Z-line, was used to identify initial cDNA clones. Additional overlapping clones were isolated and sequenced yielding a 5.4 kb contig. The encoded polypeptide contains 16 Type-II domains and four unique intervening segments. Poly-clonal sera, raised against an expressed protein fragment encoded by the 5′ end of the contig, strongly stained the Z-line of myofibrils of different species. However, the sequence of this fragment is 83% identical at the amino acid level with the previously reported C-terminal (i.e. M-line) end of chicken embryonic skeletal muscle titin. The expressed protein fragment could be phosphorylated in vitro by embryonic skeletal muscle extract and by the purified proline-directed kinase ERK1, presumably at the xSPxR recognition sites located in the first interdomain segment.
Differential distribution of two cytoplasmic variants of the α6β1 integrin laminin receptor in the ventral plasma membrane of embryonic fibroblasts
Authors: Cattelino Anna , Longhi Renato , Curtis Ivan de 
Published: April 2021

ABSTRACT
The integrin α6β1 is a receptor involved in the adhesion of several cell types to laminin. By using function-blocking antibodies, we have shown that (z60 1 is a functional laminin receptor in chick embryo fibroblasts. We also found that these cells express two variants of the α6 subunit, α6A and a6B, characterized by different cytoplasmic domains. By using indirect immunofluorescence with isoform-specific polyclonal antibodies, we showed that the two isoforms of the α6 subunit distribute differently on the ventral plasma membrane of these cells cultured on laminin-coated substrates. In fact, while the α6A subunit was found codistributing with vinculin in focal contacts, the α6B subunit showed a homogeneously distributed punctate pattern. This difference was particularly evident when preparations of ventral plasma membranes were used for the immunolocalization. Furthermore, when cells were cultured on fibronectin, a substrate not recognized by the α6β1 laminin receptor, the distribution of the two α6 isoforms was similar to that observed on laminin, with α6A still colocalizing with vinculin in focal adhesions. Our results indicate that two forms of the β6β1 laminin receptor coexpressed in the same cells show distinctive distributions, and suggest that receptor occupancy by laminin is not essential for the accumulation of the α6Aβ1 integrin in adhesion plaques.
Purification of bovine lens cell-to-cell channels composed of connexin44 and connexin50
Authors: Konig Nicola , Zampighi Guido A. 
Published: April 2021

ABSTRACT
Cell-to-cell channels composed of connexin44 and connexin50 were purified from plasma membranes of calf and fetal bovine lenses. The channels were treated with the nonionic detergents octyl-β-P-glucopyranoside and decyl-β-D-maltopyranoside, and the channel/detergent complexes purified by ion and gel filtration column chromatography. In negative staining, the channels appeared as annuli 11±0.6 nm (s.d., n=105) in diameter and as 16±0.8 nm (s.d., n=96) long particles which corresponded to top and side views of ‘complete’ cell-to-cell channels. The purified cell-to-cell channels were composed principally of a protein, called MP70, that appeared as a diffuse 55–75 kDa band in SDS-PAGE. Dephosphorylation with alkaline phosphatase transformed the diffuse 55–75 kDa band into two distinct bands of almost equal intensity. Immunoblotting showed the bands to be connexin44 and connexin50, respectively. The antibodies also recognized weaker bands composed of the unphosphorylated form of both connexins. The connexins appear to be processed independently ‘in vivo’. The unphosphorylated form of connexin50 was present in channels and membranes from fetal, calf and adult bovine lenses, while unphosphorylated connexin44 only in channels purified from fetal lenses. Therefore, lens cell-to-cell channels are composed principally of equal amounts of phosphorylated connexins 44 and 50 that appear to be assembled in the same channel (‘hybrid’).