bluuebunny/crossref_metadata_2025_split huggingface dataset:
Liver-regulating protein (LRP) is a plasma membrane protein involved in cell contact-mediated regulation of Sertoli cell function by primary spermatocytes
Authors: Gérard Nadine , Corlu Anne , Kneip Bernard , Kercret Henri , Rissel Maryvonne , Guguen-Guillouzo Christiane , Jégou Bernard 
Published: April 2021

ABSTRACT
We have identified a liver-regulating protein involved in cell contact-mediated regulation of Sertoli cell function by primary spermatocytes in rat testis. Liver-regulating protein was studied using monoclonal antibody L8 prepared from rat primitive biliary epithelial cells. This molecule was located in vivo at the interface of Sertoli cells and spermatocytes, and expressed in a stage-dependent manner (expression peaked on leptotene-zygotene sperma-tocytes). In vitro, the liver-regulating protein was found on Sertoli cell, spermatocyte and early spermatid membranes. Immunoaffinity procedures revealed two peptides of 85 and 73 kDa for Sertoli cells, while spermatocytes and sper-matids displayed a single smaller peptide of 56 kDa. The involvement of the liver-regulating protein in cell interac-tion-mediated regulation of Sertoli cell was assessed in vitro by tracing Sertoli cell transferrin and inhibin secretion, as well as mRNA synthesis in spermatocyte-Sertoli cell cocul-tures and in rat liver biliary epithelial cell-Sertoli cell cocultures, performed in the presence or absence of monoclonal antibody L8. Inhibition of the spermatocyte-and liver biliary epithelial cell-stimulated secretion of transferrin and inhibin by Sertoli cells was observed in the presence of antibody, whereas spermatocyte adhesiveness was unchanged. Using northern blot analysis, the steady state levels of transferrin mRNA decreased when the anti-liver-regulating protein antibody was added to the Sertoli cell-spermatocyte cocultures or to the Sertoli cell-liver biliary epithelial cell cocultures. The data demonstrate the role of the liver-regulating protein in cell-cell contact-mediated regulation of Sertoli function by primary spermatocytes and the important implications of this cell contact-dependent control in testicular activity.
Vimentin and CP49/filensin form distinct networks in the lens which are independently modulated during lens fibre cell differentiation
Authors: Sandilands Aileen , Prescott Alan R. , Carter Jane M. , Hutcheson Aileen M. , Quinlan Roy A. , Richards John , FitzGerald Paul G. 
Published: April 2021

ABSTRACT
The cells of the eye lens contain the type III intermediate filament protein vimentin, as well as two other intermediate filament proteins, CP49 and filensin. These two proteins appear to be unique to the differentiated lens fibre cell. Immunoblotting and confocal microscopy were used to describe changes which occur in these three intermediate filament proteins and the networks they form during fibre cell differentiation and maturation. The vimentin network was present in both epithelial cells and some fibre cells. Fibre cells were vimentin positive up to a specific point 2-3 mm in from the lens capsule where the vimentin signal was drastically reduced. The CP49/filensin network was not present in the undifferentiated epithelial cells but emerged in the differentiating fibre cells. This latter network exhibited a principally plasma membrane localization in younger fibre cells but became more cytoplasmic in older fibre cells. This change also occurred at a distinct point in fibre cell differentiation, much earlier than the observed loss of the vimentin network. The subcellular changes in the distributions of these cytoskeletal networks were correlated to the loss of the fibre cell nucleus, another feature of fibre cell differentiation. No correlation was found to changes in the vimentin network but nuclear loss did coincide with changes in the CP49/filensin network. Concomitant with nuclear pyknosis, there were also changes in the nuclear lamina as well as infringement of the nuclear compartment by CP49, as shown by confocal microscopy. This study demonstrates vimentin and the CP49/filensin network to be independent in the lens but both networks undergo dramatic changes in subcellular distribution during the differentiation/maturation of the fibre cell. Only changes in the CP49/filensin network can be correlated to nuclear loss. Thus in the lens, unlike mammalian erythropoiesis which is also characterized by nuclear loss, the vimentin network does not appear linked to nuclear retention.
Heparin-binding vitronectin up-regulates latent TGF-β production by bovine aortic endothelial cells
Authors: Ribeiro Solange M. F. , Schultz-Cherry Stacey , Murphy-Ullrich Joanne E. 
Published: April 2021

ABSTRACT
Vitronectin, a serum and extracellular matrix protein, is present in vivo in two different conformations: a native form, which does not bind heparin, and a heparin-binding conformer, which results from interactions of native vitronectin with either the thrombin-antithrombin III complex or the terminal complement complex, C5b-9. We found that vitronectin stimulates the activity of the growth regulatory peptide, TGF-β, in the conditioned media of bovine aortic endothelial cells as a result of increased production of latent TGF-β. This effect is specific for the denatured, heparin-binding, form of vitronectin, since native vitronectin has no effect on the production of latent TGF-β by those cells. Stimulation is time and concentration-dependent, but is independent of protease activity. Stimulation is dependent on the presence of cells, since there was no increase in TGF-β activity observed when vit-ronectin was added to the conditioned media after removal from cells. Furthermore, incubation of recombinant latent
TGF-β with vitronectin in a cell-free system does not result in increased TGF-β activity. Assays of total TGF-β levels in heat-treated conditioned media showed that vitronectin treatment elevates the levels of total TGF-β in the conditioned media. These results were further confirmed by western blot analysis of the conditioned media with anti-bodies specific for latent TGF-β. These data suggest that vitronectin regulates expression and/or secretion of TGF-β by bovine aortic endothelial cells. This cellular response to the heparin-binding form of vitronectin seems to be mediated by αvβ3 integrins. Since in vivo situations in which heparin-binding vitronectin is present correlate well with those in which TGF-β activity is increased, we propose that vitronectin may be a significant regulator of TGF-β activity in vivo.
Substratum surface topography alters cell shape and regulates fibronectin mRNA level, mRNA stability, secretion and assembly in human fibroblasts
Authors: Chou Laisheng , Firth James D. , Uitto Veli-Jukka , Brunette Donald M. 
Published: April 2021

ABSTRACT
The regulation of cell shape, fibronectin mRNA level, secretion and assembly by substratum surface topography was investigated in early passage human gingival fibroblasts cultured on titanium-coated smooth or V-shaped grooved substrata produced by micromachining. Cells on grooved surfaces were significantly elongated and orientated along the grooves of the substratum, while cell height, measured using confocal scanning laser microscopy, was ∼1.5-fold greater than that of cells on smooth surfaces. Northern hybridization analysis revealed that on a per cell basis the grooved surface increased the amounts of fibronectin mRNA/cell ∼3.5-fold at 16 hours, ∼1.9-fold at 40 hours and ∼2.2-fold at 90 hours, while the mRNA levels of the house-keeping gene glyceraldehyde-3-phosphate dehy-drogenase (GAPD) were constant. The amounts of secreted fibronectin on the grooved surface were increased ∼2-fold for all time points. The stability of fibronectin mRNA was also altered by substratum surface topography. The half-life of fibronectin mRNA on smooth surfaces was estimated to be ∼5 hours, but on the grooved surfaces the half-life of fibronectin mRNA showed a two-phase response: a rapid 60% reduction in the first half-life (tG ∼2 hours) and a 2.4-fold increase in the second half-life (tG ∼12 hours) relative to that observed on the smooth surface. The GAPD mRNA half-lives were essentially unaffected by the surface topography of the substrata. The grooved surface was also found to alter the amount of fibronectin assembled into the extracellular matrix, producing a ∼2-fold increase in the cultures at all time points. It thus appears that substratum surface topography alters cell shape and modulates fibronectin at the transcriptional and post-transcriptional levels, as well as the amount of fibronectin assembled into extracellular matrix. Micromachining, which has the ability to precisely control surface topography over a wide range of dimensions and shapes, appears to be a useful technique in investigating the relationship between cell shape and function.
Fibronectin fibril growth in the extracellular matrix of the Xenopus embryo
Authors: Winklbauer Rudolf , Stoltz Cornelia 
Published: April 2021

ABSTRACT
We show that the mechanism of fibronectin fibril formation on the blastocoel roof of the Xenopus embryo is comparable to that in other systems. Fibril assembly is inhibited by RGD peptide, by an amino-terminal fragment of fibronectin, and by cytochalasin B. When added exogeneously, intact fibronectin, but not a 110 kDa cell binding fragment of fibronectin, is incorporated into fibrils. Thus, the blastocoel roof of Xenopus represents a valid model system for the study of fibronectin fibril formation in situ. Moreover, we show that fibril formation can be induced experimentally in this system. Examination of fibril elongation by double-labelling experiments reveals that individual, unbranched fibronectin fibrils grow only at one end, i.e. in a unipolar fashion. The rate of elongation is 4.7 μm/min. Most fibrils grow only for a short time, and the increase in total fibril length per cell is driven by the repeated initiation of new fibrils. Assembly of fibronectin into fibrils precedes cross-linking of fibronectin into multimers in this system.
The GTPase Rab3a is associated with large dense core vesicles in bovine chromaffin cells and rat PC12 cells
Authors: Darchen François , Senyshyn Jan , Brondyk William H. , Taatjes Douglas J. , Holz Ronald W. , Henry Jean-Pierre , Denizot Jean-Pierre , Macara Ian G. 
Published: April 2021

ABSTRACT
Small GTPases of the rab family control intracellular vesicle traffic in eukaryotic cells. Although the molecular mechanisms underlying the activity of the Rab proteins have not been elucidated yet, it is known that the function of these proteins is dependent on their precise subcellular localization. It has been suggested that Rab3a, which is mainly expressed in neural and endocrine cells, might regulate exocytosis. Recently, direct experimental evidence supporting this hypothesis has been obtained. Consistent with such a role for Rab3a in regulated exocytosis was the previously reported specific association of Rab3a with synaptic vesicles and with secretory granules in adrenal chromaffin cells. Since the latter result, based on subcellular fractionation, has been controversial, we have re-investigated the subcellular localization of this GTP-binding protein by using a combination of morphological techniques. Bovine chromaffin cells were labelled with an affinity-purified polyclonal anti-Rab3a antibody and analyzed by confocal microcopy. Rab3a was found to colocalize partially with dopamine β-hydroxylase, a chromaffin granule marker. In agreement with this observation, immunoelectron microscopy revealed a specific staining of chromaffin granules. In addition to large dense core vesicles, some small vesicles were labelled. To eliminate the possibility that the staining was due to a Rab3a-related protein, we investigated by immunoelectron microscopy the localization of an epitope-tagged Rab3a expressed in rat PC12 cells. Secretory granules were specifically labelled, whereas clear microvesicles were not. These results provide further evidence supporting a specific association of the GTPase Rab3a with large dense core secretory vesicles.
Epidermal growth factor induces tyrosine phosphorylation and reorganization of the tight junction protein ZO-1 in A431 cells
Authors: Van Itallie Christina M. , Balda Maria S. , Anderson James Melvin 
Published: April 2021

ABSTRACT
Addition of epidermal growth factor (EGF) to A431 human epidermal carcinoma cells results in actin reorganization and phosphorylation of several cytoskeletal proteins. In the present study, we found that EGF treatment of this cell line also results in the redistribution and tyrosine phosphorylation of ZO-1. In normal polarized epithelial cells, ZO-1 is restricted to the cytoplasmic surface of the most apical of the intercellular junctions, the tight junction. In contrast, ZO-1 in the majority of unstimulated A431 cells in small subconfluent islands colocalizes with actin along the lateral cell membranes and in rare microspikes and membrane ruffles. Exposure to EGF results in a transient redistribution of actin into an apically positioned ring. ZO-1 becomes highly focused at apical sites of cell contact and co-localizes with the newly formed band of perijunctional actin. Coincidently, ZO-1 and another tight junction protein, ZO-2, become transiently phosphorylated on tyrosine residues, as determined by anti-phosphotyrosine immunoblotting. Pre-treatment of A431 cells with cytochalasin D, which disrupts normal microfilament organization, does not affect EGF-dependent phosphorylation of the EGF receptor. However, cytochalasin D pretreatment blocks both the EGF-induced ZO-1 rearrangement and tyrosine phosphorylation, suggesting that these responses are dependent on an intact actin microfilament system. We speculate that the transient tyrosine phosphorylation of ZO-1 in response to EGF treatment may be involved in remodeling of intercellular junctions in A431 cells.
Hormone-induced meiotic maturation in Xenopus oocytes occurs independently of p70s6k activation and is associated with enhanced initiation factor (eIF)-4F phosphorylation and complex formation
Authors: Morley Simon J. , Pain Virginia M. 
Published: April 2021

ABSTRACT
Hormone-induced meiotic maturation of the Xenopus oocyte is regulated by complex changes in protein phosphorylation. It is accompanied by a stimulation in the rate of translation, manifest at the level of polypeptide chain initiation. At later times in the maturation process, this reflects an increased ability for mRNA to interact with the 40 S ribosomal subunit. In mammalian cells there is growing evidence for the regulation of translation by phosphorylation of ribosomal protein S6 and of initiation factors responsible for the binding of mRNA to ribosomes. In this report, we show that although the 70 kDa form of S6 kinase is activated within 1.5 hours in response to progesterone or insulin, a time critical for protein synthesis, its activation is not required for hormone-induced stimulation of translation rates or maturation. In response to progesterone, activation of translation occurs in parallel with enhanced phosphate labelling of eIF-4α and eIF-4γ and eIF-4F complex formation, events which are thought to facilitate the interaction of eIF-4F with the mRNA cap structure. However, with insulin, activation of translation occurs prior to detectable de novo phosphorylation of eIF-4F, although a small enhancement of turnover of phosphate on eIF-4α may occur at this early time. With either hormone, enhanced phosphate labelling of eIF-4α is shown to reflect activation of eIF-4α kinase(s), which co-incides temporally with activation of p42 MAP and p90rsk kinases. The possible role of initiation factor modification on increased translation rates during meiotic maturation is discussed.
Trimeric G proteins regulate the cytosol-induced redistribution of Golgi enzymes into the endoplasmic reticulum
Authors: Hidalgo Josefina , Muñiz Manuel , Velasco Angel 
Published: April 2021

ABSTRACT
Streptolysin O-permeabilized cells incubated with a high concentration (5-10 mg/ml) of cytosolic proteins and ATP-generating system exhibit redistribution into the endoplasmic reticulum (ER) of Golgi integral proteins (mannosidase II, galactosyltransferase, TGN 38), detected by immuno-fluorescence. In addition, mannosidase II is detected in the ER of cells exposed to a high concentration of cytosolic proteins and processed for immunolectron microscopy by immunoperoxidase. The redistribution process requires ATP and is not affected by previous microtubule depolymerization. Ultrastructural observations indicate that Golgi disassembly occurs by budding of coated vesicles. This stage of the process is inhibited by GTPγS, AlF(3-5), transducin βγ subunits, and mastoparan, indicating the involvement of trimeric G proteins. At a later stage, vesicles lose their coats and fuse with the ER. This part of the process does not occur in cells incubated at either 15°C or 20°C, or exposed to N-ethylmaleimide. In cells treated with either cholera or pertussis toxin Golgi redistribution into the ER shows a 50-fold lower requirement for cytosolic factors than in untreated cells. These data suggest a regulatory role for both αs and αi trimeric G proteins in the normal Golgi-ER retrograde transport taking place in intact cells.
Characterisation of a desmocollin isoform (bovine dsc3) exclusively expressed in lower layers of stratified epithelia
Authors: Yue K. K. M. , Holton J. L. , Clarke J. P. , Hyam J. L. M. , Hashimoto T. , Chidgey M. A. J. , Garrod D. R. 
Published: April 2021

ABSTRACT
Desmocollins are cadherin-like glycoproteins involved in cell adhesion and plaque formation in desmosome junctions. Three distinct isoforms, the products of different genes, have been found in bovine tissues. We have reported previously that one of these, DSC3, is expressed only in basal and lower suprabasal layers of stratified epithelia. Using RT-PCR we have now obtained the complete cDNA coding sequence of mature bovine DSC3. It has alternatively spliced ‘a’ and ‘b’ forms found in other desmocollins but is unique in having a 43 instead of a 46 base pair exon. We have characterised a monoclonal antibody, 07-4G, which is specific for the Dsc3 protein, recognising an epitope in the extracellular domain. Immunofluorescent staining with 07-4G confirms that this isoform is found only in stratified epithelia, being strongly expressed in the basal cell layers of these tissues. The intensity of expression fades gradually in the suprabasal layers and disappears completely below the upper limit of desmosome expression. These results suggest that Dsc3 plays an important role in cell epithelial differentiation.
Immunomorphological characterization and effects of 12-(s)-hete on a dynamic intracellular pool of the αiibβ3-integrin in melanoma cells
Authors: Tímár J. , Bazaz R. , Kimler V. , Haddad M. , Tang D. G. , Robertson D. , Tovari J. , Taylor J. D. , Honn K. V. 
Published: April 2021

ABSTRACT
In metastatic B16a murine melanoma cells, αIIbβ3 integrin was shown to be one of the key adhesion molecules responsible for matrix adhesion and spreading. Upon stimulation, αIIbβ3 can be upregulated at the cell surface due to translocation of the receptor to the plasma membrane from an intracellular pool. Here we have characterized this integrin pool as a tubulovesicular structure (TVS) corresponding to endosomes. TVS was found to be associated temporarily with microtubules and intermediate filaments especially after protein kinase C (PKC) stimulation with a lipoxygenase metabolite of arachidonic acid, 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE]. After PKC stimulation, the predominantly vesicular TVS became elongated and αIIbβ3 appeared at the apical plasma membrane and microvilli. Disruption of either the microtubules or intermediate filaments prevented the 12-(S)-HETE effect both on vesicular to tubular transition of TVS as well as on surface expression of this integrin. The connection with the Golgi system of the integrin-containing TVS was proved by a Golgi-inhibitor (brefeldin A) pretreatment, which prevented the PKC-stimulation-induced TVS elongation and subsequent receptor-upregulation at the cell surface. After a soluble ligand binding (mAb to the αIIbβ3 complex) the surface receptor endocytosed back to the TVS indicating the presence of a dynamic, cytoskeleton associated integrin pool in melanoma cells.
Apoptosis precedes necrosis of dystrophin-deficient muscle
Authors: Tidball James G. , Albrecht Douglas E. , Lokensgard Beth E. , Spencer Melissa J. 
Published: April 2021

ABSTRACT
The current view that death of dystrophin-deficient muscle fibers is a necrotic process relies primarily upon the histological appearance of the tissue after the degenerative process is well advanced. Here, we tested this view by examining the possibility that apoptosis is a component of dystrophin-deficient muscle cell death. Three assays for apoptosis were employed in analyzing prenecrotic, peak necrotic and regenerated hindlimb muscle of mdx mice: (1) terminal deoxynucleotidyl transferase (TdT) mediated end-labeling of DNA in nuclei in tissue sections; (2) assays for DNA ladders; and (3) electron microscopic assays for the presence of organelles undergoing structural changes characteristic of apoptosis. At all ages sampled, mdx muscle contained apoptotic nuclei, according to TdT-mediated dUTP labeling of tissue sections. Nuclei in regenerated mdx muscle fibers did not display apoptosis. dUTP-labeled nuclei in control C57 muscles were rare or absent at all ages sampled. DNA from 4-week-old mdx mice was found to be cleaved into fragments indicative of preferential cleavage at internucleosomal sites. Electron microscopic analysis showed that organelle structural changes indicating apoptosis appear before pathological changes diagnostic of necrosis. For example, condensed mitochondria, fragmented sarcoplasmic reticulum and nuclei with chromatin condensations resembling apoptosis appear in fibers that otherwise possess normal morphology. Together, the findings show that apoptosis precedes any detectable necrotic change in mdx muscle, and that apoptotic events continue into the stage of dystrophic pathology that is currently viewed as necrosis. Thus, apoptosis characterizes the onset of pathology in dystrophin-deficient muscle which is followed secondarily by necrotic processes.