bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a PC12 cell sub-clone (PC12-c41) with enhanced neurite outgrowth capacity: implications for a modulatory role of high molecular weight tau in neuritogenesis
Authors: Teng Kenneth K. , Georgieff Irene S. , Aletta John M. , Nunez Jacques , Shelanski Michael L. , Greene Lloyd A. 
Published: April 2021

ABSTRACT
To address the means by which diversity of neuronal morphology is generated, we have isolated and characterized naturally occurring variants of rat PC12 pheochromocytoma cells that exhibit altered neurite outgrowth properties in response to nerve growth factor (NGF). We describe here a PC12 cell sub-clone, designated PC12-clone 41 (PC12-C41), that displays significant increases in neurite abundance and stability when compared with the parental line. This difference does not appear to be due to an altered sensitivity or responsiveness to NGF or to a more rapid rate of neurite extension. Because of the role of the cytoskeleton in neuritogenesis, we examined a panel of the major cytoskeletal proteins (MAP 1.2/1B, -tubulin, chartins, peripherin, and high and low molecular weight (HMW and LMW) taus) whose levels and/or extent of phosphorylation are regulated by NGF in PC12 cultures. Although most cytoskeletal proteins showed little difference between PC12 and PC12-C41 cells (± NGF treatment), there was a significant contrast between the two lines with respect to tau expression. In particular, while NGF increases the total specific levels of tau in both cell types to similar extents (by about twofold), the proportion comprising HMW tau is threefold higher in the PC12-C41 clone than in PC12 cells. A comparable difference was observed under substratum conditions that were non- permissive for neurite outgrowth and so this effect was not merely a consequence of the differential neuritogenic capacities of the two lines. The distinction between the expression of HMW and LMW taus in PC12 and PC12-C41 cells (± NGF) was also observed at the level of the messages encoding these proteins. Such findings indicate that initiation of neurite outgrowth in PC12 cultures does not require a massive induction of tau expression and raise the possibility that HMW and LMW taus may have differential capacities for modulating neuronal morphology.
Incorporation of tubulin subunits into dimers requires gtp hydrolysis
Authors: Fontalba Ana , Paciucci Rosanna , Avila Jesus , Zabala Juan C. 
Published: April 2021

ABSTRACT
A toroid multisubunit complex of 800-900 kDa has been implicated in assisting protein folding of at least two cytoplasmic proteins, actin and tubulin. This process is dependent on the presence of magnesium ions and ATP hydrolysis. In vitro translation of cDNAs encoding different α- and β-tubulin isotypes also gives rise to the formation of complexes of about 300 kDa. These complexes have been functionally implicated in the incorporation of tubulin monomers within the tubulin heterodimer. This work shows that, in addition to ATP hydrolysis, the incorporation of newly synthesized tubulin subunits into functional heterodimers requires GTP hydrolysis in the presence of magnesium ions. A two-step process is suggested, a first ATP-dependent step in which the 900 kDa complexes are implicated in a similar way to the step taking place in actin folding, and a second GTP-dependent step in which the 300 kDa complexes are involved in the assembly of the heterodimer.
Inhibition of constitutive protein secretion from lactating mouse mammary epithelial cells by FIL (feedback inhibitor of lactation), a secreted milk protein
Authors: Rennison Michelle E. , Kerr Marian , Addey Caroline V. P. , Handel Susan E. , Turner Mark D. , Wilde Colin J. , Burgoyne Robert D. 
Published: April 2021

ABSTRACT
The effect of a protein feedback inhibitor of lactation (FIL) on casein synthesis and secretion was examined using isolated acini from lactating mouse mammary gland. As previously found, FIL partially inhibited protein synthesis but produced an additional inhibition of constitutive casein secretion. The inhibition of synthesis and secretion showed similar dose-dependency and the inhibition was fully reversible. Constitutive secretion of pre-formed protein was inhibited by FIL in a pulsechase protocol, indicating that the inhibitor regulated protein secretion by reducing protein movement through the secretory pathway independently of any initial inhibition of synthesis. Regulated exocytosis was not inhibited since casein release due to elevation of cytosolic Ca2+ concentration by the ionophore ionomycin was unaffected. Brefeldin A, which is known to block ER-to-Golgi transport, also inhibited both protein synthesis and secretion in mammary cells. The action of FIL on synthesis and secretion and previously described actions on casein degradation would be consistent with a block at an early stage in the secretory pathway. In support of this idea FIL treatment was found to result in vesiculation and swelling of the endoplasmic reticulum. These data provide evidence for a novel control of a constitutive secretory pathway by a physiological extracellular regulatory protein.
Involvement of a phosphoprotein on the zymogen granule membrane in the control of regulated exocytosis in the exocrine pancreas
Authors: MacLean Carol M. , Marciniak Stefan J. , Hall Diane V. , Edwardson J. Michael 
Published: April 2021

ABSTRACT
The pancreatic acinar cell is one of a number of cell types in which phosphoproteins are believed to be involved in the control of regulated exocytosis. We have examined the effects of three agents that affect secretion in the acinar cell on the phosphorylation states of proteins on the zymogen granule membrane. We show that Ca2+ and GTP S, which stimulate secretion, also stimulate the phosphorylation of a protein of Mr 45,000 (p45) on isolated zymogen granules. On the other hand, the protein kinase inhibitor genistein inhibits both secretion and phosphorylation of p45. For all three agents, p45 phosphorylation is affected over concentration ranges identical to those that affect secretion. The stimulatory effect of GTP S and the inhibitory effect of genistein are also seen when the phosphorylation state of p45 on granules within permeabilized cells is examined. Ca2+, however, over the same concentration range, now causes dephosphorylation of p45. Furthermore, the time-course of this effect is similar to that of Ca2+-triggered secretion. Phosphorylation of p45 is exclusively on serine, with no detectable phosphorylation on either threonine or tyrosine. We propose that exocytosis in pancreatic acini is controlled at least in part through the phosphorylation/dephosphorylation of p45, with dephosphorylation acting as a trigger for exocytosis.
Brain-type creatine kinase in photoreceptor cell outer segments: role of a phosphocreatine circuit in outer segment energy metabolism and phototransduction
Authors: Hemmer Wolfram , Riesinger Ingrid , Wallimann Theo , Eppenberger Hans M. , Quest Andrew F. G. 
Published: April 2021

ABSTRACT
Different isoforms of creatine kinase, an important enzyme of vertebrate energy metabolism, were localized in bovine photoreceptor cells, with particular emphasis on the identification and quantification of the brain-type isoform within the outer segment compartment. Using immunofluorescence and immunoelectron microscopy, brain-type creatine kinase was shown to be present in bovine photoreceptor cell outer and inner segments. The presence of this isoenzyme in rod outer segments was additionally confirmed by immunoblotting and immunolabeling of isolated rod outer segments. The content of creatine kinase in rod outer segments was quantified by measuring creatine kinase activity after membrane disruption with detergent. The ATP regeneration potential provided by the creatine kinase in isolated, washed bovine rod outer segments was 1.2 ± (0.4) i.u. mg 1 rhodopsin. This value was calculated to be at least an order of magnitude larger than that necessary to replenish the energy required for cGMP resynthesis in rod outer segments, and high enough to regenerate the entire ATP pool of rod outer segments within the time span of a photic cycle.
A mitochondrial creatine kinase isoenzyme was located within the ellipsoid portions of bovine rod and cone inner segments by immunofluorescence microscopy and, using immunogold staining, was specifically localized in the mitochondria clustered within bovine rod and cone inner segments. These results suggest that vertebrate photoreceptor cells contain a functional phos-phocreatine circuit. Outer segment creatine kinase may play an important role in phototransduction by providing energy for the visual cycle, maintaining high local ATP/ADP ratios and consuming protons produced by enzymes located in the outer segment.
Localisation of granulocyte macrophage colony-stimulating factor in human long-term bone marrow cultures Biological and immunocytochemical characterisation
Authors: Wynter E. de , Allen T. , Coutinho L. , Flavell D. , Flavell S. U. , Dexter T. M. 
Published: April 2021

ABSTRACT
The distribution of granulocyte macrophage colony-stimulating factor (GM-CSF) in human long-term bone marrow cultures (HLTBMC) was examined using two monoclonal antibodies raised using purified recombinant GM-CSF and a third commercially available GM-CSF antibody. The antibodies were able to bind to purified recombinant GM-CSF and showed inhibition of GM-CFC colonies in the presence of both recombinant and native protein. All antibodies displayed similar pat-terns of distribution in both permeabilised and non-per-meabilised stromal cell preparations. Fibroblasts were labelled at their periphery in early cultures and both endothelial cells and fibroblasts showed cytoplasmic labelling with anti-GM-CSF. The fact that GM-CSF appears to be sequestered by cells of the bone marrow stroma raises the possibility that it is synthesised by these cells and may regulate activity of the progenitor cells in the haemopoietic foci. In contrast, early progenitor cells within the foci did not stain with any of the anti-GM-CSF antibodies. Adipocytes, which differenti-ate from fibroblasts in these cultures, showed a diffuse staining pattern. Two types of macrophage staining were observed in the non-permeabilised cells; those exhibiting only autofluorescence and those that bound the antibody. Intracellular staining was apparent in a small sub-population. Generally, the staining persisted up to eight weeks of culture and thereafter declined, becoming virtually undetectable after 12 weeks. This correlates with the pattern of GM-CFC production in long-term bone marrow cultures.
Antisense RNA inactivation of gp138 gene expression results in repression of sexual cell fusion in Dictyostelium discoideum
Authors: Fang Hui , Higa Kazuhiro Aiba, Mikihiko , Urushihara Hideko , Yanagisawa Kaichiro 
Published: April 2021

ABSTRACT
A glycoprotein, gp138, is implicated in the sexual cell fusion of Dictyostelium discoideum. We previously cloned and sequenced the two genes encoding the gp138 protein, GP138A and GP138B (Fang et al. (1993)Dev. Biol. 156, 201-208). Here, we have constructed a vector producing antisense RNA for the gp138 genes and have transformed the vector into Dictyostelium cells. The transformed cells showed a reduction in the amounts of gp138 mRNA and protein and their sexual cell fusion activity was considerably repressed.
The small GTP-binding protein rab6p is redistributed in the cytosol by brefeldin A
Authors: Roa Michèle , Cornet Véronique , Yang Chun-Zhi , Goud Bruno 
Published: April 2021

ABSTRACT
Rab6 protein belongs to the Sec4/Ypt/rab subfamily of small GTP-binding proteins involved in intracellular membrane trafficking in yeast and mammalian cells. Its localization both in medial and trans-Golgi network prompted us to study the effects of brefeldin A (BFA) on rab6p redistribution. By two techniques, indirect immunofluorescence and cell fractionation, we investigated the fate of rab6p and compared it to other Golgi or trans-Golgi network markers in BHK-21 and NIH-3T3 cells. BFA, at 5 g/ml, induced redistribution of rab6p according to a biphasic process: during the first 10-15 minutes, tubulo-vesicular structures – colabelled with a bona fide medial Golgi marker called CTR 433-were observed; these structures were then replaced by punctate diffuse staining, which was stable for up to 3 hours. The 110 kDa peripheral membrane protein β-COP was released much more rapidly from the Golgi membranes, whereas the trans-Golgi network marker TGN 38 relocated to the microtubule organizing center.
The kinetics of reversion of BFA action on these anti-gens was also followed by immunofluorescence. Consis-tent with these results, rab6 antigen, originally found as 40% in the cytosolic versus 60% in the particulate (P 150,000 g) fraction, became almost entirely cytosolic; moreover, it partitioned in the aqueous phase of Triton X-114 whereas the membrane fraction was detergent-soluble. Rab6p did not become part of the coatomers after its BFA-induced release from Golgi structures. Three requirements seemed to be necessary for such a release: integrity of the microtubules, presence of energy, and a hypothetical trimeric G protein, as revealed by the respective roles of nocodazole, ATP depletion, and sensitivity to aluminium fluoride. Finally, we have shown that BFA does not prevent attachment of newly synthesized rab6p to membranes.
Immunoisolation of Kex2p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single Golgi compartment
Authors: Bryant Nia J. , Boyd Alan 
Published: April 2021

ABSTRACT
One of the Golgi compartments of Saccharomyces cere - visiae is defined by the presence of a specific endopro-teinase, Kex2p, which cleaves precursor polypeptides at pairs of basic residues. We have used antibodies directed against the cytoplasmically disposed C-terminal domain of Kex2p to develop an immuno-affinity procedure for the isolation of Kex2p-containing organelles. The method gives a high yield of sealed organelles that are essentially free of contamination from other secretory pathway organelles while being significantly enriched for two other late Golgi enzymes, dipeptidylaminopeptidase A and the Kex1 carboxypeptidase. Our findings provide clear evidence for a single yeast Golgi com-partment containing all three late-processing enzymes, which is likely to be the functional equivalent in yeast of the mammalian trans-Golgi network.
The phosphotyrosine phosphatase inhibitor vanadyl hydroperoxide induces morphological alterations, cytoskeletal rearrangements and increased adhesiveness in rat neutrophil leucocytes
Authors: Bennett Paul A. , Dixon Richard J. , Kellie Stuart 
Published: April 2021

ABSTRACT
The functional consequences of treating rat neutrophils with the potent tyrosine phosphatase inhibitor vanadyl hydroperoxide (pervanadate) has been investigated. Pervanadate induced rapid increases in cellular protein phosphotyrosine content in a dose-dependent manner. This treatment also resulted in a change in morphology of the cells from a rounded to a polarised morphology, with many cells exhibiting uropods, pseudopodia and increased membrane activity. Pervanadate induced a transient actin polymerisation and reorganisation similar to that in agonist-stimulated cells. The pervanadate-induced increases in tyrosine phosphorylation, shape change and actin polymerisation were inhibited by the tyrosine kinase inhibitors tyrphostin and erbstatin, indicating that these phenomena were mediated by the constitutive activity of cellular tyrosine kinases. Double flu-orescence experiments demonstrated that there was a co-localisation of tyrosine phosphorylated proteins with F-actin in both pervanadate- and agonist-stimulated neutrophils. Pervanadate also induced spreading of neutrophils on tissue culture substrata with concurrent changes in F-actin localisation including unusual F-actin-containing structures. These results demonstrate that morphological changes and cytoskeletal reorganisation in neutrophils are regulated by tyrosine phosphorylation, and that inhibition of tyrosine phosphatase activity in neutrophils is sufficient to activate motile machinery of these cells. These results suggest that an alternative pathway involved in neutrophil stimulation might be via inhibition of endogenous tyrosine phosphatases rather than activation of tyrosine kinases.
Co-localization of class II antigen and exogenous antigen in the rat enterocyte
Authors: Gonnella Patricia A. , Wilmore Douglas W. 
Published: April 2021

ABSTRACT
The role of class II major histocompatibility antigens in classical antigen-presenting cells has been described (Unanue (1984)Annu. Rev. Immunol. 2, 395-428; Watts and McConnell (1987)Rev. Immunol. 5, 461-475). Whether enterocytes, which also express class II antigens, can act as antigen-presenting cells in vivo is not known. One pre-requisite for a role for enterocytes in antigen presentation is an interaction between exogenous antigen and class II antigens. Our results demonstrate that class II antigen and exogenous antigen absorbed from the gastrointestinal tract are co-localized within endocytic compartments and along the basolateral membranes of enterocytes.
The locomotion, shape and pseudopodial dynamics of unstimulated Dictyostelium cells are not random
Authors: Killich Thomas , Plath Peter J. , Wei Xiang , Bultmann Heino , Rensing Ludger , Vicker Michael G. 
Published: April 2021

ABSTRACT
The dynamic periphery of unstimulated, preaggregation, hunger-stage Dictyostelium discoideum amoebae was investigated by time-lapse videomicroscopy and digital image processing. Circular maps (i.e. of each of 360 radii around the cell transformed upon Cartesian coordinates) were constructed around the centroid of individual cell images and analysed in time series. This novel technique generated spatiotemporal structures of various degrees of order in the maps, which resemble classical wave interference patterns. The patterns thus demonstrate that cell movement is not random and that cells are intrinsically vibrating bodies, transited by self-organized, superpositioned, harmonic modes of rotating oscillatory waves (ROWS). These waves appear to depend upon spatiotemporal oscillations in the physico-chemical reactions associated with actin polymerization, and they govern pseudopodial movements, cell shape and locomotion generally. ROWS in this case are unrelated to the cyclic-AMP-regulated oscillations, which characterize later, aggregative populations of Dictyostelium. However, the exposure of aggregation-stage cells to a pulse of the chemoattractant cyclic-AMP induces a characteristic sequence of changes in the global cellular concentration and spatiotemporal distri-bution of fibrillar (F-)actin. This reaction begins with what appears to be a phase resetting of ROWS and it may, therefore, underlie the cellular perception of and response to chemotactic signals. We also develop here an analytical mathematical description of ROWS, and use it to simulate cell movements accurately.
Formation of hemidesmosomes in cells of a transformed murine mammary tumor cell line and mechanisms involved in adherence of these cells to laminin and kalinin
Authors: Sonnenberg A. , de Melker A. A. , de Velasco A. M. Martinez , Janssen H. , Calafat J. , Niessen C. M. 
Published: April 2021

ABSTRACT
Keratinocytes attach to an underlying basement mem-brane by adhesion junctions called hemidesmosomes. We have characterized a cell line, RAC-11P/SD, estab-lished from a murine mammary tumor, which differen-tiates into squamous epithelium and forms well defined hemidesmosomes. These hemidesmosomes contain the integrin 6 4 as well as the hemidesmosomal plaque proteins BP230 and HD1 and are associated with a matrix containing kalinin and laminin. We examined how these cells adhere to laminin and to kalinin present in matrices as well as immunopurified kalinin. We show that adhesion to laminin is energy dependent but does not require an intact actin-containing cytoskeleton. The affinity for kalinin proved to be greater and binding to kalinin was still observed when cells had been treated with deoxyglucose and azide to inhibit metabolic energy. Binding to laminin (or fragment E8), but not to kalinin was partially blocked by a monoclonal antibody specific for the integrin 6 subunit, and only in the initial phase of adhesion. The antibody efficiently blocked adhesion to laminin of cells treated with the microfilament dis-rupting drug cytochalasin B, but only partially blocked the adhesion of cytochalasin B-treated cells to kalinin, while adherence of cells treated with deoxyglucose and azide to kalinin was blocked completely. The integrin α6β4 is redistributed to the basal surface during adhe-sion and then is organized into ring-like structures when cells are bound to laminin and localized into hemidesmosomes in cells adhered to kalinin. We suggest that anti-α6 hinders the binding of theα6β4 integrins to its ligands laminin and kalinin, but cannot prevent adhe-sion when clustering of the integrin has become com-plete. In addition, there is evidence that adhesion to kalinin is mediated by a second receptor, which associ-ates with the actin-containing cytoskeleton. The pres-ence of such a second receptor is suggested because the cells can spread on kalinin, but not when they have been treated with cytochalasin B. On laminin spreading does not occur, irrespective of whether cells have been treated with cytochalasin B or not. The integrin α3β1, which has been identified as a receptor for kalinin but not for laminin, is strongly expressed in RAC-11P/SD cells and it seems likely that this integrin is responsible for spreading of cells on kalinin.
Vacuolar H+-ATPase of Dictyostelium discoideum A monoclonal antibody study
Authors: Fok Agnes K. , Clarke Margaret , Ma Le , Allen Richard D. 
Published: April 2021

ABSTRACT
A Dictyostelium membrane fraction rich in vacuolar proton pumps, previously described by Nolta et al. (J. Biol. Chem. 266, 18,318-18,323, 1991), was used as the immunogen for production of monoclonal antibodies. We obtained antibodies that recognized polypeptides of 100 kDa and 68 kDa, corresponding to the two largest subunits of the vacuolar proton pump. In indirect immunofluorescence experiments, these two subunits were located on an interconnected collection of tubules and vacuoles. On frozen thin sections they were found principally on membranes of vacuoles and collections of small vesicles typically located just internal to the plasma membrane. These vesicles and vacuoles had elec-tron-translucent lumens. No other structures in axeni-cally grown Dictyostelium cells were labeled to a signif-icant extent by these two antibodies. Using an affinity-purified antibody to calmodulin and a mono-specific antibody to the B subunit of the chromaffin granule vacuolar ATPase, markers known to label the membranes of the contractile vacuole complex in Dictyostelium (Zhu and Clarke, J. Cell Biol. 118, 347-358, 1992; Heuser et al., J. Cell Biol. 121, 1311-1327, 1993), we showed that the 100 kDa and 68 kDa subunits had the same distribution as these two markers. Co-local-ization was seen in both interphase and mitotic cells. Thus, our results support the conclusion that vacuolar proton pumps are located principally on the membranes of the contractile vacuole complex in Dictyostelium. In addition, in indirect immunofluorescence experiments, these monoclonal antibodies provided improved images of the organization of the contractile vacuole system.