bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tau interacts with src-family non-receptor tyrosine kinases
Authors: Lee Gloria , Newman S. Todd , Gard David L. , Band Hamid , Panchamoorthy Govindaswamy 
Published: April 2021

ABSTRACT
Tau and other microtubule-associated proteins promote the assembly and stabilization of neuronal microtubules. While each microtubule-associated protein has distinct properties, their in vivo roles remain largely unknown. Tau is important in neurite outgrowth and axonal development. Recently, we showed that the amino-terminal region of tau, which is not involved in microtubule interactions, is important in NGF induced neurite outgrowth in PC12 cells. Here we report that a proline rich sequence in the amino terminus of tau interacts with the SH3 domains of fyn and src non-receptor tyrosine kinases. Tau and fyn were co-immunoprecipitated from human neuroblastoma cells and co-localization of tau and fyn was visualized in co-transfected NIH3T3 cells. Co-transfection of tau and fyn also resulted in an alteration in NIH3T3 cell morphology, consistent with an in vivo interaction. Fyn-dependent tyrosine phosphorylation of tau occurred in transfected cells and tyrosine phosphorylated tau was identified in human neuroblastoma cells as well. Our data suggest that tau is involved in signal transduction pathways. An interaction between tau and fyn may serve as a mechanism by which extracellular signals influence the spatial distribution of microtubules. The tyrosine phosphorylation of tau by fyn may also have a role in neuropathogenesis, as fyn is upregulated in Alzheimer’s disease.
Cytoplasmic calcium gradients and calmodulin in the early development of the fucoid alga Pelvetia compressa
Authors: Pu Rongsun , Robinson Kenneth R. 
Published: April 2021

ABSTRACT
The predicted existence of cytoplasmic Ca2+gradients during the photopolarization of the zygotes of the brown algae, Pelvetia and Fucus, has proved to be difficult to establish, and the downstream targets of the putative gradients are not known. We have used quantitative microinjection of the long excitation wavelength Ca2+indicator, Calcium Crimson, and of antibodies against calmodulin to investigate these matters in the zygotes and early embryos of Pelvetia.
We found that there is a window of cytoplasmic Calcium Crimson concentration that gives an adequate signal above autofluorescence yet allows normal development of the zygotes. As Calcium Crimson is not a ratiometric indicator, we injected other zygotes with a Ca2+-insensitive dye, rhodamine B, and imaged the cells at the same time that Calcium Crimson-injected cells were imaged. Ratios were calculated by dividing the averaged pixel values of Calcium Crimson images by the averaged pixel values of corresponding rhodamine B images. By this method, we observed the formation of a cytoplasmic Ca2+ gradient within one hour of the exposure of the cells to unilateral blue light during the photosensitive period. The region of high Ca2+was localized to and predictive of the site of future rhizoid formation. We validated this somewhat indirect method by applying it to the growing rhizoid, where the existence of a tip-localized Ca2+gradient is well established. The method clearly revealed the known gradient.
The injection of ungerminated zygotes with antibodies made against Dictyostelium calmodulin inhibited germination, and this inhibition was abolished if the calmodulin antibodies were coinjected with an excess of purified maize calmodulin. Likewise, the growth of the rhizoids was inhibited by calmodulin antibody injections. The fungus-derived calmodulin antagonist, ophiobolin A, which has previously been shown to be a potent inhibitor of germination, also inhibited rhizoidal growth. Our results provide evidence that a cytoplasmic Ca2+gradient is present during photopolarization and that calmodulin acts as a mediator of Ca2+gradients throughout the early developmental processes of germination and rhizoidal growth in Pelvetia compressa.
Axonal outgrowth of cultured neurons is not limited by growth cone competition
Authors: Lamoureux Phillip , Buxbaum Robert E. , Heidemann Steven R. 
Published: April 2021

ABSTRACT
We have examined the question of scarcity-driven competition for outgrowth among growth cones of a single neuron. We measured spontaneous neurite elongation rates from 85 hours of videotape of the arbors of 31 chick sensory neurons in culture. These rate measurements were analyzed in ten minute periods that allowed cell bodies to be classified as to the number of their growth cones and the elongation to be analyzed as a series of discrete events. Comparing periods in which neurons maintained simple bipolar morphology we find no temporal competition between the two growth cones. That is, periods of above-average growth by one growth cone are not compensated by below-average growth during the same period by its sibling growth cone. Analyzing all outgrowth from a neuron based on its number of growth cones shows that net elongation rate from a single cell body is a linear function of the number of growth cones from 1 to 11. These observations suggest that growth cones behave independently and are not limited by availability of structural precursors. A surplus pool of structural precursors available for normal growth is also indicated by the high capacity for growth from single neurites when experimentally stimulated by mechanical tension. In addition, towing one or more neurites at above average rates does not cause any decline in simultaneous growth cone-mediated outgrowth from a single neuron compared to the 2-3 hour period prior to experimentally induced elongation. This high capacity for growth combined with the often observed, intermittant growth behavior of individual growth cones suggests that neurite outgrowth is intrinsically limited primarily by poor growth cone ‘performance,’ not scarcity-driven competition. We postulate that growth cones are poor ‘tractors,’ exerting too little tension to exploit the available capacity for axonal elongation.
Proteolytic processing and cell biological functions of the amyloid precursor protein
Authors: De Strooper Bart , Annaert Wim 
Published: April 2021

ABSTRACT
Recent research has identified some key players involved in the proteolytic processing of amyloid precursor protein (APP) to amyloid β-peptide, the principal component of the amyloid plaques in Alzheimer patients. Interesting parallels exists with the proteolysis of other proteins involved in cell differentiation, cholesterol homeostasis and stress responses. Since the cytoplasmic domain of APP is anchored to a complex protein network that might function in axonal elongation, dendritic arborisation and neuronal cell migration, the proteolysis of APP might be critically involved in intracellular signalling events.
Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process
Authors: Coverley Dawn , Pelizon Cristina , Trewick Sarah , Laskey Ronald A. 
Published: April 2021

ABSTRACT
Cdc6 is essential for the initiation of DNA replication in all organisms in which it has been studied. In addition, recombinant Cdc6 can stimulate initiation in G1 nuclei in vitro. We have analysed the behaviour of recombinant Cdc6 in mammalian cell extracts under in vitro replication conditions. We find that Cdc6 is imported into the nucleus in G1 phase, where it binds to chromatin and remains relatively stable. In S phase, exogenous Cdc6 is destroyed in a process that requires import into the nucleus and phosphorylation by a chromatin-bound protein kinase. Recombinant cyclin A-cdk2 can completely substitute for the nucleus in promoting destruction of soluble Xenopus and human Cdc6.
Despite this regulated destruction, endogenous Cdc6 persists in the nucleus after initiation, although the amount falls. Cdc6 levels remain constant in G2 then fall again before mitosis. We propose that cyclin A-cdk2 phosphorylation results in destruction of any Cdc6 not assembled into replication complexes, but that assembled proteins remain, in the phosphorylated state, in the nucleus. This process could contribute to the prevention of reinitiation in human cells by making free Cdc6 unavailable for re-assembly into replication complexes after G1 phase.
Fa1p is a 171 kDa protein essential for axonemal microtubule severing in Chlamydomonas
Authors: Finst Rip J. , Kim Peter J. , Griffis Eric R. , Quarmby Lynne M. 
Published: April 2021

ABSTRACT
A key event in deflagellation or deciliation is the severing of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. Previous genetic analysis revealed three genes that are essential for deflagellation in Chlamydomonas. We have now identified the first of these products, Fa1p, a protein required for Ca2+-dependent, axonemal microtubule severing. Genetic mapping and the availability of a tagged allele allowed us to physically map the gene to the centromere-proximal domain of the mating-type locus. We identified clones of Chlamydomonas genomic DNA that rescued the Ca2+-dependent axonemal microtubule severing defect of fa1 mutants. The FA1 cDNA, obtained by RT-PCR, encodes a novel protein of 171 kDa, which is predicted to contain an amino-terminal coiled-coil domain and three Ca2+/calmodulin binding domains. By western analysis and subcellular fractionation, the FA1 product is enriched in flagellar-basal body complexes. Based on these observations and previous studies, we hypothesize that a Ca2+-activated, Ca2+-binding protein binds Fa1p leading ultimately to the activation of axonemal microtubule severing.
Genetic defects in acetylcholine signalling promote protein degradation in muscle cells of Caenorhabditis elegans
Authors: Szewczyk Nathaniel J. , Hartman James J. , Barmada Sami J. , Jacobson Lewis A. 
Published: April 2021

ABSTRACT
A myosin-lacZ fusion, expressed in 103 muscle cells of Caenorhabditis elegans, reports on how proteolysis in muscle is controlled by neural and intramuscular signals. Upon acute starvation, the fusion protein is degraded in the posterior 63 cells of the body-wall muscle, but remains stable in 32 anterior body-wall muscles and 8 vulval muscle cells. This distinction correlates with differences in the innervation of these cells. Reporter protein in the head and vulval muscles becomes labile upon genetic ‘denervation’ in mutants that have blocks in pre-synaptic synthesis or release of acetylcholine (ACh) or post-synaptic reception at nicotinic ACh receptors (nAChR), whereas protein in all 103 muscles is stabilized by the nicotinic agonist levamisole in the absence of ACh production. Levamisole does not stabilize muscle protein in nAChR mutants that are behaviorally resistant to levamisole. Neural inputs thus exert negative control over the proteolytic process in muscle by stimulating muscle nicotinic ACh receptors.
The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2)
Authors: Litman Thomas , Brangi Mariafiorella , Hudson Eric , Fetsch Patricia , Abati Andrea , Ross Douglas D. , Miyake Keisuke , Resau James H. , Bates Susan E. 
Published: April 2021

ABSTRACT
Mechanisms of drug resistance other than P-glycoprotein are of increasing interest as the list of newly identified members of the ABC transport family has grown. We sought to characterize the phenotype of the newly discovered ABC transporter encoded by the mitoxantrone resistance gene, MXR, also known as ABCP1 or BCRP. The pharmacodynamics of mitoxantrone and 12 other fluorescent drugs were evaluated by confocal microscopy in four multidrug-resistant human colon (S1) and breast (MCF-7) cancer cell lines. We utilized two sublines, MCF-7 AdVp3000 and S1-M1-80, and detected overexpression of MXR by PCR, immunoblot assay and immunohistochemistry. These MXR overexpressing sublines were compared to cell lines with P-glycoprotein- and MRP-mediated resistance. High levels of cross-resistance were observed for mitoxantrone, the anthracyclines, bisantrene and topotecan. Reduced levels of mitoxantrone, daunorubicin, bisantrene, topotecan, rhodamine 123 and prazosin were observed in the two sublines with high MXR expression. Neither the P-glycoprotein substrates vinblastine, paclitaxel, verapamil and calcein-AM, nor the MRP substrate calcein, were extruded from MCF-7 AdVp3000 and S1-M1-80 cells. Thus, the multidrug-resistant phenotype due to MXR expression is overlapping with, but distinct from, that due to P-glycoprotein. Further, cells that overexpress the MXR protein seem to be more resistant to mitoxantrone and topotecan than cells with P-glycoprotein-mediated multidrug resistance. Our studies suggest that the ABC half-transporter, MXR, is a potent, new mechanism for conferring multiple drug resistance. Definition of its mechanism of transport and its role in clinical oncology is required.
The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia
Authors: Chew Catherine S. , Parente John A. , Chen Xunsheng , Chaponnier Christine , Cameron Richard S. 
Published: April 2021

ABSTRACT
Lasp-1 is a unique LIM and src homology 3 (SH3) domain-containing protein that was initially identified as a 40 kDa cAMP-dependent phosphoprotein in the HCl-secreting gastric parietal cell. Because cAMP is a potent stimulator of parietal cell acid secretion, we have hypothesized that changes in lasp-1 phosphorylation might be involved in the regulation of ion transport-related activities, perhaps by modulating interactions among cytoskeletal and/or vesicle-associated proteins. In this study, we demonstrate that the cAMP-dependent acid secretory agonist, histamine, induces a rapid, sustained rise in parietal cell lasp-1 phosphorylation and this increase in phosphorylation is closely correlated with the acid secretory response. In addition, elevation of intracellular cAMP concentrations appear to induce a partial redistribution of lasp-1 from the cell cortex, where it predominates along with the γ-isoform of actin in unstimulated cells, to the β-actin enriched, apically-directed intracellular canalicular region, which is the site of active proton transport in the parietal cell. Additional studies demonstrate that although lasp-1 mRNA and protein are expressed in a wide range of tissues, the expression is specific for certain actin-rich cell types present within these tissues. For example, gastric chief cells, which contain relatively little F-actin and secrete the enzyme, pepsinogen, by regulated exocytosis, do not appear to express lasp-1. Similarly, lasp-1 was not detected in pancreatic acinar cells, which secrete enzymes by similar mechanisms and also contain relatively low levels of F-actin. Lasp-1 also was not detectable in proximal tubules in the kidney, in gastrointestinal smooth muscle, heart or skeletal muscle. In contrast, expression was prominent in the cortical regions of ion-transporting duct cells in the pancreas and in the salivary parotid gland as well as in certain F-actin-rich cells in the distal tubule/collecting duct. Interestingly, moderate levels of expression were also detected in podocytes present in renal glomeruli and in vascular endothelium. In primary cultures of gastric fibroblasts, lasp-1 was present mainly within the tips of lamellipodia and at the leading edges of membrane ruffles. Taken together these results support the hypothesis that the lasp-1 plays an important role in the regulation of dynamic actin-based, cytoskeletal activities. Agonist-dependent changes in lasp-1 phosphorylation may also serve to regulate actin-associated ion transport activities, not only in the parietal cell but also in certain other F-actin-rich secretory epithelial cell types.
Colocalization of chaperone Cpn60, proinsulin and convertase PC1 within immature secretory granules of insulin-secreting cells suggests a role for Cpn60 in insulin processing
Authors: Arias Ariel E. , Vélez-Granell Carlos S. , Mayer Gaétan , Bendayan Moïse 
Published: April 2021

ABSTRACT
Many of the mechanisms that control insulin processing and packaging by interaction with different elements along the secretory pathway remain poorly understood. We have investigated the possibility that Cpn60, a member of the heat shock protein family, may be present in rat insulin- secreting cells, participating in the proinsulin-insulin maturation process. Immunofluorescence and high resolution immunocytochemical studies revealed the presence of the Cpn60 protein all along the insulin secretory pathway, being particularly abundant over the proinsulin-containing immature secretory granules. Double-labeling experiments showed associations between Cpn60 and proinsulin, as well as between Cpn60 and PC1 convertase, with a preferential binding to proinsulin. These findings paralleled those of coimmunoprecipitation studies showing the Cpn60 chaperone and the mature form of the PC1 convertase in proinsulin immunoprecipitates, as well as the PC1 in Cpn60 immunoprecipitates from total islet cell extracts. In vitro binding of Cpn60 to proinsulin, insulin and glucagon was also documented. Cpn60, significantly abundant in proinsulin-containing secretory granules where conversion of proinsulin to insulin takes place, and the colocalization of the chaperone with proinsulin and PC1 convertase suggest that the Cpn60 protein may play a role directing precise molecular interactions during insulin processing and/or packaging.
Retroviral expression of Wnt-1 and Wnt-7b produces different effects in mouse mammary epithelium
Authors: Naylor Stuart , Smalley Matthew J. , Robertson David , Gusterson Barry A. , Edwards Paul A. W. , Dale Trevor C. 
Published: April 2021

ABSTRACT
Several Wnt genes are expressed in the postnatal mouse mammary gland and are thought to be involved in mammary gland development. Ectopic expression of Wnt-1, which is not normally expressed in the mammary gland, drives the formation of a pre-neoplastic hyperplasia. Cell culture-based assays have shown that Wnt-1 and some mammary-expressed Wnts transform C57MG cells. This has led to the suggestion that Wnt-1 functions as an oncogene through the inappropriate activation of developmental events that are normally controlled by the ‘transforming’ class of Wnts. In this study, Wnt-7b was expressed in vivo using recombinant retroviruses. Wnt-7b did not alter normal mammary gland development despite having similar effects to Wnt-1 in cell culture. We conclude that the in vitro classification of Wnts as ‘transforming’ does not correlate with the transformation in vivo. To facilitate the analysis of Wnt-expression, a lacZ-containing, bicistronic recombinant retrovirus was developed. Immunohistochemistry and electron microscopy identified retrovirally transduced myoepithelial and luminal epithelial cells in normal and hyperplastic tissues. The distribution of transduced cells in mammary outgrowths was consistent with current models of mammary stem cell identity.
Prostanoids mediate the protective effect of trefoil factor 3 in oxidant-induced intestinal epithelial cell injury: role of cyclooxygenase-2
Authors: Tan Xiao-Di , Chen Yi-Hua , Liu Qian-Ping , Gonzalez-Crussi Frank , Liu Xue-Li 
Published: April 2021

ABSTRACT
Trefoil factors are small peptides found in several mammalian tissues including gut, respiratory tract and brain. Their physiological function is not well understood. Among them, trefoil factor 3 (intestinal trefoil factor) is known to be cytoprotective in the gut. However, the molecular mechanism and secondary mediators of trefoil factor 3 action are not known. In the present study, we examined whether the cyclooxygenase pathway is involved in trefoil factor 3 action. We showed that trefoil factor 3 significantly induces the production of prostaglandin E2 and prostaglandin I2 in IEC-18 cells (an intestinal epithelial cell line) in a dose dependent manner. Western blot and immunohistochemistry revealed that trefoil factor 3 (2.5 μM) up-regulates the expression of cyclooxygenase-2 but not cyclooxygenase-1 in IEC-18 cells. Treating cells with trefoil factor 3 (10 μM) significantly attenuated reactive oxygen species-induced IEC-18 cell injury. This effect is blocked by NS-398 (10 μM), a selective cyclooxygenase-2 inhibitor. Moreover, we demonstrated that exogenously administered carbacyclin (1 μM, a stable analogue of prostaglandin I2) and/or prostaglandin E2 (1 μM) caused a significant reduction of reactive oxygen species-induced cell injury, mimicking the effect of trefoil factor 3. In summary, our results indicate that trefoil factor 3 activates cyclooxygenase-2 in intestinal epithelium to produce prostaglandin I2 and prostaglandin E2, which function as survival factors and mediate the cytoprotective action of trefoil factor 3 against oxidant injury.
Relative locations of the centromere and imprinted SNRPN gene within chromosome 15 territories during the cell cycle in HL60 cells
Authors: Nogami Masahiro , Kohda Atsushi , Taguchi Hiroshi , Nakao Mitsuyoshi , Ikemura Toshimichi , Okumura Katsuzumi 
Published: April 2021

ABSTRACT
Investigations of imprinted regions provide clues that increase our understanding of the regulation of gene functions at higher order chromosomal domains. Here, the relative positions of the chromosome 15 centromere and the imprinted SNRPN gene in interphase nuclei of human myeloid leukemia HL60 cells were compared, because the homologous association of this imprinted chromosomal domain was previously observed in lymphocytes and lymphoblasts. Four targets including the chromosome 15 territory, its centromere, the SNRPN gene on this chromosome, and the nucleus, were visualized simultaneously in three-dimensionally preserved nuclei using multicolor fluorescence in situ hybridization, and the spatial distributions of these probes were analyzed with a cooled CCD camera deconvolution system. We found that preferential association of SNRPN interhomologues did not occur during the cell cycle in HL60 cells, although this gene exhibited asynchronous replication and monoallelic expression in this cells. SNRPN was found to localize at the periphery of the chromosome territories, and it preferentially faced the nuclear membrane, unlike the adjacent centromeric repeat. The SNRPN gene and the centromere were located close to each other late in S phase, reflecting that these DNA segments may be compacted into the same intranuclear subcompartments with the progress of S phase and in course of preparation for the following G2 phase. Our results suggest that, although an imprinted gene has features similar to those observed with intranuclear localization of other gene coding sequences, the characteristic of mutual recognition of imprinted regions is determined by certain cellular regulation, and it is not necessary for the allele-specific features of an imprinted gene.