bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cellular localization of RNA14p and RNA15p, two yeast proteins involved in mRNA stability
Authors: Bonneaud Nathalie , Minvielle-Sebastia Lionel , Cullin Christophe , Lacroute François 
Published: April 2021

ABSTRACT
RNA14 and RNA15 were originally identified by temperature-sensitive mutations that cause a rapid decrease in poly(A)-tail length and overall mRNA levels at the restrictive temperature. We have raised antibodies to the RNA14 and RNA15 proteins, and used subcellular fractionation and immunofluorescence to localize these proteins within the yeast cell. RNA14p is a 73 kDa protein found in both the nucleus and the cytoplasm, whilst RNA15p is a 42 kDa protein detected only in the nucleus. The observation that both proteins are found in the nucleus is in agreement with previous genetic data which suggest an interaction between RNA14p and RNA15p. Also the joint nuclear localization is consistent with the biochemical data suggesting a role in polyadenylation. The detection of significant amounts of RNA14p in the cytoplasm opens the possibility of a second function for this protein, either in cytoplasmic regulation of mRNA deadenylation or, more interestingly, in mRNA stability.
Brefeldin A causes structural and functional alterations of the trans-Golgi network of MDCK cells
Authors: Wagner Martina , Rajasekaran Ayyappan K. , Hanzel David K. , Mayor Satyajit , Rodriguez-Boulan Enrique 
Published: April 2021

ABSTRACT
The trans-Golgi network (TGN) of MDCK cells is exquisitely sensitive to the fungal metabolite brefeldin A (BFA), in contrast to the refractory Golgi stack of these cells. At a concentration of 1 μg/ml, BFA promoted extensive tubulation of the TGN while the medial Golgi marker α-mannosidase II was not affected. Tubules emerging minutes after addition of the drug contained both the apical marker influenza hemagglutinin (HA), previously accumulated at 20°C, and the fusion protein interleukin receptor/TGN38 (TGG), a TGN marker that recycles basolaterally, indicating that, in contrast to TGN vesicles, TGN-derived tubules cannot sort apical and basolateral proteins. After 60 minutes treatment with BFA, HA and TGG tubules formed extensive networks widely spread throughout the cell, different from the focused centrosomal localization previously described in non-polarized cells. The TGG network partially codistributed with an early endosomal tubular network loaded with transferrin, suggesting that the TGG and endosomal networks had fused or that TGG had entered the endosomal network via surface recycling and endocytosis. The extensive structural alterations of the TGN were accompanied by functional disruptions, such as the extensive mis-sorting of influenza HA, and by the release of the TGN marker γ-adaptin. Our results suggest the involvement of BFA-sensitive adaptor proteins in TGNrsurface transport.
Conformation dependence of integrin-type II collagen binding: Inability of collagen peptides to support α2β1 binding, and mediation of adhesion to denatured collagen by a novel α5β1-fibronectin bridge
Authors: Tuckwell Danny S. , Ayad Shirley , Grant Michael E. , Takigawa Masaharu , Humphries Martin J. 
Published: April 2021

ABSTRACT
The mechanism of interaction of chondrocytic cells with cartilage-specific type II collagen has been examined using HCS-2/8 human chondrosarcoma cells as a model system. By the criteria of specific collagen secretion and integrin expression profile, HCS-2/8 have a similar differentiated phenotype to normal chondrocytes and are therefore a good model system. HCS-2/8 cells were able to attach and spread on both native and heat-denatured pepsinised type II collagen, and assays using denatured cyanogen bromide fragments apparently localised the major cell binding site to the CB10 fragment. However, when they were used as soluble inhibitors, cyanogen bromide fragments were found to block adhesion to denatured collagen, but had no effect on either attachment or spreading on the native molecule. The inability of cyanogen bromide fragments to reproduce the cell binding site of native collagen demonstrated a strict dependence on collagen conformation. This was also reflected in the receptors that were employed by HCS-2/8 cells for binding to type II collagen: binding to native collagen was mediated by the integrin α2β1 while binding to denatured collagen was mediated by a novel α5β1-fibronectin bridge. The identification of this bridge adds to the mechanisms by which cells can bind to denatured collagens. The previously characterised KDGEA active site peptide from type I collagen was found to be inactive as an inhibitor of type II collagen-mediated adhesion. The implications of these findings for the strategies used to identify adhesive active sites within collagens are discussed. In particular, these data suggest that, unlike other integrin ligands, a synthetic peptide-based approach is not suitable for the identification of collagen active sites.
Intracellular free calcium oscillations in normal and cleavage-blocked embryos and artificially activated eggs of Xenopus laevis
Authors: Keating Thomas J. , Cork R. John , Robinson Kenneth R. 
Published: April 2021

ABSTRACT
We have measured levels of intracellular free calcium ([Ca2+]i) in albino Xenopus laevis embryos using recombinant aequorin and a photon-counting system. We observed sinusoidal oscillations in [Ca2+]i that had the same frequency as cleavage, with cleavage occurring when [Ca2+]i was lowest. An increase in calcium was seen to precede first cleavage. The cyclic changes in calcium were superimposed on a secondary pattern that increased, peaked between third and fifth cleavages and then slowly declined to a level similar to that measured before first cleavage. The amplitude of the oscillations was small during the first few cleavages but became larger with each cycle, with the largest oscillations occurring when the secondary pattern peaked (between third and fifth cleavage). As the secondary pattern declined, the amplitude of the oscillations also became smaller. The oscillations are due to release of calcium from intracellular stores, since the signal was the same in calcium-free solution as in normal medium.
When cleavage was blocked with the microtubule-disrupting drugs colchicine or nocodazole, the [Ca2+]i oscillations persisted. Calcium oscillations of a similar magnitude and frequency were also present in artificially activated eggs. The secondary pattern was different in cleavage-blocked embryos and artificially activated eggs, the baseline increasing until about the third cycle and then remaining elevated for the rest of the recording (&gt;8 hours).
By fixing embryos at various points in the calcium cycle, we determined that mitosis began shortly after calcium levels reached their peak and was complete before the calcium level dropped to its lowest point. The fact that the calcium oscillations persist when nuclear division is not occurring suggests that either they operate independently of any downstream events that they might control or they are related to other cyclic activities in the eggs and embryos such as cycling of pHi, MPF, or surface contraction waves.
When aequorin was injected into individual blastomeres of 64-cell embryos, the shape of the signal was substantially different from that seen in recordings from whole embryos. Compared to the whole-embryo recordings, the signal from a subregion of the embryo rose more quickly and had a slower, biphasic decline. These differences indicate that [Ca2+]i increases are not occurring uniformly across the embryo but are spatially localized, perhaps progressing as waves.
Band 6 protein, a major constituent of desmosomes from stratified epithelia, is a novel member of the armadillo multigene family
Authors: Hatzfeld Mechthild , Kristjansson Gunnar Ingi , Plessmann Uwe , Weber Klaus 
Published: April 2021

ABSTRACT
Desmosomes are intercellular adhering junctions characteristic of epithelial cells. Several constitutive proteins-desmoplakin, plakoglobin and the transmembrane glycoproteins desmoglein and desmocollin-have been identified as fundamental constituents of desmosomes in all tissues. A number of additional and cell type-specific constituents also contribute to desmosomal plaque formation. Among these proteins is the band 6 polypeptide (B6P). This positively charged, non-glycosylated protein is a major constituent of the plaque in stratified and complex glandular epithelia. Using an overlay assay we show that purified keratins bind in vitro to B6P. Thus B6P may play a role in ordering intermediate filament networks of adjacent epithelial cells. To characterize the structure of B6P in the desmosome we have isolated cDNA clones representing the entire coding sequence. The predicted amino acid sequence of human B6P shows strong sequence homology with a murine p120 protein, which is a substrate of protein tyrosine kinase receptors and of p60v-rc. P120 and B6P show amino-terminal domains differing distinctly in length and sequence. These are followed in both proteins by 460 residues that display a series of imperfect repeats corresponding to the repeats in the cadherin binding proteins armadillo, plakoglobin and P-catenin. Over this repeat region B6P and p120 share 33% sequence identity (54% similarity). These sequence characteristics define B6P as a novel member of the armadillo multigene family and raise the question of whether the structural proteins B6P, plako-globin, β-catenin and armadillo share some function. Since armadillo, plakoglobin, β-catenin and p120 seem involved in signal transduction this may also hold for B6P. The amino-terminal region of B6P (residues 1 to 263) shows no significant homology to any known protein sequence. It may therefore be involved in unique functions of B6P.
Differential calmodulin binding to three myosin-1 isoforms from liver
Authors: Coluccio Lynne M. 
Published: April 2021

ABSTRACT
We have previously purified and characterized two myosin-1 isoforms from rat liver (molecular masses 130 kDa and 110 kDa; L. M. Coluccio and C. Conaty (1993) Cell Motil. Cytoskel. 24, 189–199). Here, we describe the purification and characterization from liver of a third myosin-1 (molecular mass 105 kDa) and determine the number of calmodulin molecules associated with each of these three myosin-1 isoforms. The 105 kDa polypeptide, solubilized from liver homogenates with the addition of ATP, co-sediments with F-actin, co-purifies with calmod-ulin, and binds calmodulin in the presence of EGTA. Anti-bodies directed against chicken intestinal brush border myosin-1 cross-react with the 105 kDa polypeptide on immunoblots. Partial peptide sequence analysis indicates that the polypeptide corresponds with an MM1γ gene product that represents a myosin-1 isoform cloned from mouse brain (Sherr et al. (1993)J. Cell Biol. 120, 1405-1416). A comparison of calmodulin binding to the now three isolated forms of myosin-1 in liver shows that in solution the 105 kDa and 110 kDa polypeptides bind two molecules of calmodulin each whereas the 130 kDa binds six molecules of calmodulin.
Ultraviolet radiation-induced melanogenesis in human melanocytes Effects of modulating protein kinase C
Authors: Carsberg C. J. , Warenius H. M. , Friedmann P. S. 
Published: April 2021

ABSTRACT
The mechanism by which ultraviolet radiation induces melanogenesis in epidermal melanocytes is unknown. Previous observations that in cultured human melanocytes 1-oleoyl-2-acetylglycerol augmented both basal and ultra-violet radiation-induced melanogenesis, suggested that the responses were mediated via protein kinase C. However, paradoxically the phorbol ester TPA was without effect. Therefore, the present study has examined the involvement of protein kinase C in melanogenesis. Analysis of the isozyme profile of human melanocytes revealed the presence of protein kinase C α, βI, ε and ζ but not the isozyme η. Following exposure to 500 nM TPA for 24 hours, isozymes α, βI and ε were downregulated, but ζ was unaffected. Similar isozyme profiles were observed in S91 and SKMEL3 melanoma cells. The melanogenic responses to 1-oleoyl-2-acetylglycerol and ultraviolet radiation were unaffected by inhibition of protein kinase C with Ro31-8220, or ablation by downregulation with 500 nM TPA, in human melanocytes and melanoma cells. 1-Oleoyl-2-acetyl-glycerol had no effect on protein kinase C activity in human melanocytes, as measured by rapid phosphorylation of the 80 kDa protein myristoylated alanine-rich C kinase substrate (MARCKS). Ultraviolet radiation induced a small increase in MARCKS protein phosphorylation but this effect was inhibited by pretreatment for 24 hours with 500 nM TPA, which had no effect on ultraviolet-induced melanogenesis. Overall, these findings indicate that 1-oleoyl-2-acetylglycerol and ultraviolet radiation activate melanogenesis via protein kinase C-independent pathways.
The heparin-binding domain of heparin-binding EGF-like growth factor can target Pseudomonas exotoxin to kill cells exclusively through heparan sulfate proteoglycans
Authors: Mesri Enrique A. , Ono Minoru , Kreitman Robert J. , Klagsbrun Michael , Pastan Ira 
Published: April 2021

SUMMARY
Heparin-binding EGF-like growth factor (HB-EGF) is a smooth muscle cell mitogen composed of both EGF receptor and heparin-binding domains. To better understand the function of its domains, intact HB-EGF or its heparin-binding (HB) domain (amino acids 1-45) were fused to a mutant Pseudomonas exotoxin with an inactivated cell-binding domain. The resulting chimeric toxins, HB-EGF-PE* and HB-PE*, were tested on tumor cells, proliferating smooth muscle cells and a mutant Chinese hamster ovary cell line deficient in heparan sulfate proteoglycans (HSPGs). Two targets were found for HB-EGF-PE*. Cells were killed mainly through EGF receptors, but the HB domain was responsible for killing via HSPGs. HB-PE* did not bind to the EGF receptor and thus was cytotoxic by interacting exclusively with HSPGs. We conclude that the HB domain of HB-EGF is able to mediate internalization through HSPGs, without requiring the EGF receptor.
Yeast homolog of mammalian mitogen-activated protein kinase, FUS3/DAC2 kinase, is required both for cell fusion and for G1 arrest of the cell cycle and morphological changes by the CDC37 mutation
Authors: Fujimura Hiro-aki 
Published: April 2021

ABSTRACT
Saccharomyces cerevisiae FUS3/DAC2 protein kinase, a homolog of mammalian mitogen-activated protein (MAP) kinase, inactivates a G1 cyclin encoded by the CLN3 gene to arrest cell division in the G1 phase and activates a tran-scriptional factor STE12 in response to mating pheromone during sexual conjugation. To elucidate the role of the FUS3/DAC2 gene product in the mating process, I con-structed and characterized dac2 cln3 double mutants. Here, I show that FUS3/DAC2 is required for completion of cell fusion even in the dac2 cln3 double mutants in which the pheromone response is restored, suggesting that FUS3/DAC2 plays a positive role in cell fusion during con-jugation. In addition, the cdc dac2 and cdc37 ste double mutants were constructed and investigated for their phe-notypes to clarify the relationship between FUS3/DAC2, STE7 or STE11 and CDC gene products (CDC28, 36, 37 and 39). The results indicate that FUS3/DAC2 may act upstream of CDC28 and provide evidence that the G1 arrest and morphological changes conferred by the cdc37 mutation may require FUS3/DAC2 (MAP kinase), STE7(MEK) and STE11 (MEK kinase).
The regulated degradation of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase reporter construct occurs in the endoplasmic reticulum
Authors: Lecureux Lloyd W. , Wattenberg Binks W. 
Published: April 2021

ABSTRACT
The rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase, is regulated at a number of levels. One important mechanism is regulation of the half-life of the protein by a controlled proteolytic system. This comes about in response to downstream products of the sterol biosynthetic pathway. Little is known about this system, including where in the cell this regulated degradation occurs. HMG CoA reductase resides in the endoplasmic reticulum. To localize the site of regulated degradation of HMG CoA reductase, we used a construct that fuses the N-terminal membrane-anchoring domain of HMG CoA reductase in-frame with β-galactosidase as a reporter domain (HM-Gal). HM-Gal has previously been shown to reproduce faithfully the degradative properties of native HMG CoA reductase (Chun et al. (1990)J. Biol. Chem. 265, 22004-22010). CHO cells transfected with DNA encoding HM-Gal were exposed to mevalonic acid, which enhances the rate of HMG CoA reductase degradation several fold, and leads to the reduction of the steady state levels of HM-Gal by 80-90%. To accumulate HMG CoA reductase at the site of degradation, cells were simultaneously treated with N-acetyl-leucyl-leucyl-norleucinal (ALLN), which inhibits the protease responsible for reductase degradation. HM-Gal was localized morphologically by immunofluorescence and biochemically by measuring β-galactosidase activity in Percoll gradients of cellular homogenates. Using either technique HM-Gal localization was indistinguishable from that of ER markers in both control cells and in cells treated to accumulate HMG CoA reductase at the site of degradation. We conclude that the regulated degradation of HMG CoA reductase occurs in the ER or an ER-like compartment.
Telomere length and proliferation potential of hematopoietic stem cells
Authors: Lansdorp Peter M. 
Published: April 2021

ABSTRACT
Hematopoietic stem cells have typically been defined as pluripotent cells with self-renewal capacity. Recent studies have shown striking differences in the mean length of telomeric repeat sequences at the end of chromosomes from human hematopoietic cells at different stages of development. The most likely explanation for these observations is that hematopoietic stem cells, like all other somatic cells studied to date, lose telomeric DNA upon each cell division. In this review, limitations in the replicative potential of hematopoietic stem cells are discussed in the context of possible clinical use of such cells for transplantation and gene therapy.
Osteogenic protein-1 promotes growth and maturation of chick sternal chondrocytes in serum-free cultures
Authors: Chen Ping , Vukicevic Slobodan , Sampath T. Kuber , Luyten Frank P. 
Published: April 2021

ABSTRACT
We examined the effect of recombinant human osteogenic protein-1 (OP-1, or bone morphogenetic protein-7), a member of the bone morphogenetic protein family, on growth and maturation of day 11, 15 and 17 chick sternal chondrocytes in high density monolayers, suspension and agarose cultures for up to 5 weeks. OP-1 dose-dependently (10-50 ng/ml) promoted chondrocyte maturation associated with enhanced alkaline phosphatase activity, and increased mRNA levels and protein synthesis of type X collagen in both the presence and absence of serum. In serum-free conditions, OP-1 promoted cell proliferation and chondrocyte maturation, without requiring either thyroid hormone or insulin, agents known to support chick chondrocyte differentiation in vitro. When grown in agarose under the same conditions, TGF-β1 and retinoic acid neither initiated nor promoted chondrocyte differentiation. The results demonstrate that OP-1, as the sole medium supplement, supports the maturation of embryonic chick sternal chondrocytes in vitro.
Three-dimensional co-location of RNA polymerase I and DNA during interphase and mitosis by confocal microscopy
Authors: Gilbert N. , Lucas L. , Klein C. , Menager M. , Bonnet N. , Ploton D. 
Published: April 2021

ABSTRACT
The relative three-dimensional co-location of RNA polymerase I (RPI) and DNA was studied using confocal laser scanning microscopy during interphase and all the steps of mitosis in human cancerous cells.
For each step of the cell cycle, immunolabeled RPI molecules and DNA specifically stained with chromomycin A3 were simultaneously imaged at high resolution through numerous optical sections. Then, all the data obtained were used to generate transverse sections, anaglyphs and volumic representations, which are all prerequisite approaches to a representative study of the three-dimensional organization of the nucleolus and the mitotic chromosomes. Our results indicated that in the interphasic nuclei, in which DNA is organized as a regular 3-D network, RPI was present within numerous irregular spheres arranged as several twisted necklaces. During metaphase, RPI labeling was segregated into pairs of spheres and typical crescent-shaped structures; both were centrally located within the set of chromosomes. During anaphase and telophase, a typical central and symmetric arrangement of labeled structures was systematically seen among the decondensing chromosomes, arranged as a regular cylinder and as a hollow half-sphere, respectively. This typical 3-D organization of structures containing RPI relative to DNA is another strong example of the non-random organization of the genome during interphase and mitosis.
Direct involvement of a lamin-B-related (54 kDa) protein in the association of intermediate filaments with the postsynaptic membrane of the Torpedo marmorata electrocyte
Authors: Cartaud Annie , Jasmin Bernard J. , Changeux Jean-Pierre , Cartaud Jean 
Published: April 2021

ABSTRACT
Mechanisms by which motor innervation induces postsynaptic membrane differentiation and functional compart-mentalization of the subneural sarcoplasm in skeletal muscle fibres are still poorly understood. However, trans-membrane control of cytoskeletal activities by the nerve terminal may be considered. Here, we examine several properties of a 54 kDa protein, previously identified in the postsynaptic membrane of the Torpedo marmorata electrocyte with anti-lamin B antibodies, in order to study its role in the assembly of the subneural intermediate filament meshwork. Using a ligand blot assay, we show that this protein binds desmin, a type III intermediate filaments protein, at micromolar concentrations. Moreover, purified acetylcholine receptor-rich membrane fragments are able to generate arrays of desmin filaments in vitro. Immunofluorescence experiments indicate that the 54 kDa protein becomes associated with the acetylcholine receptor-rich membrane at an early stage of development of the electrocyte, and that a polarized desmin network develops concomitantly from the postsynaptic membrane. Taken together, these data show that, like karyoskeletal lamin B, the 54 kDa protein is involved in the organization of the subneural intermediate filament meshwork. Control of the assembly of the subneural cytoskeleton by components of the postsynaptic membrane may thus be a prerequisite for the functional compartmentalization of the muscle fibre triggered by motor innervation.