bluuebunny/crossref_metadata_2025_split huggingface dataset:
Suppression of apoptosis by v-ABL protein tyrosine kinase is associated with nuclear translocation and activation of protein kinase C in an interleukin-3-dependent haemopoietic cell line
Authors: Evans Caroline A. , Lord Janet M. , Owen-Lynch P. Jane , Johnson Gerald , Dive Caroline , Whetton Anthony D. 
Published: April 2021

ABSTRACT
We previously demonstrated that activation of v-ABL protein tyrosine kinase resulted in suppression of apoptosis following interleukin-3 removal using an interleukin-3-dependent haemopoietic cell line transfected with a temperature-sensitive mutant of the v-abl oncoprotein (IC.DP). Cellular signalling events associated with the activation of v-ABL included increased levels of sn-1,2-diacylglycerol, an activator of protein kinase C. Calphostin C, a PKC inhibitor, restored apoptosis to interleukin-3-deprived IC.DP cells expressing active v-ABL. However, chronic exposure to the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate to downregulate protein kinase C did not attenuate the survival of IC.DP cells expressing active v-ABL. Translocation of a classical protein kinase C isozyme(s) to the nuclear fraction was observed 6 hours after activation of v-ABL, when nuclear protein kinase C activity was increased approximately 2-fold. The protien kinase C isozyme responsible, which was only partially downregulated by 12-O-tetradecanoyl phorbol 13-acetate, was identified as protein kinase C βII. This translocation of protein kinase C βII to the nucleus was inhibited by calphostin C. Taken together, these results suggest that nuclear translocation and activation of PKCβII may play a role in v-ABL-mediated suppression of apoptosis.
Delayed cyclin A and B1 degradation in non-transformed mammalian cells
Authors: Girard Franck , Fernandez Anne , Lamb Ned 
Published: April 2021

ABSTRACT
Cyclins A and B are known to exhibit significant differences in their function, cellular distribution and timing of degradation at mitosis. On the basis of observations in marine invertebrates and Xenopus, it was proposed that cyclin destruction triggers cdc2 kinase inactivation and anaphase onset. However, this model has recently been questioned, both in Xenopus and in budding yeast.
In this report, we present evidence for delayed degradation of both cyclins A and B1 in non-transformed mammalian cells. Indeed, by means of indirect immunofluorescence and confocal microscopy, we show that cyclins A and B1 are present up to anaphase in REF52, Hs68, human primary fibroblasts and NRK epithelial cells. In marked contrast, cyclin A is shown to be degraded within metaphase and cyclin B just at the transition to anaphase in HeLa and two transformed cell lines, derivatives of normal NRK and REF52. These results further support the notion that cyclin destruction might be not correlated with anaphase onset in normal cells and highlight a significant difference in the fate of mitotic cyclins between transformed and non-transformed cells.
Gap junctions and cell polarity: Connexin32 and connexin43 expressed in polarized thyroid epithelial cells assemble into separate gap junctions, which are located in distinct regions of the lateral plasma membrane domain
Authors: Guerrier Annie , Fonlupt Pierre , Morand Isabelle , Rabilloud Rachida , Audebet Christine , Krutovskikh Vladimir , Gros Daniel , Rousset Bernard , Munari-Silem Yvonne 
Published: April 2021

ABSTRACT
Epithelial cells of the thyroid gland present an uncommon connexin expression pattern, they coexpress connexin32 and connexin43. In the present work, we have analyzed the membrane distribution of these two connexins to determine: (i) whether they co-assemble in the same gap junctions or form separate gap junctions; and (ii) whether their location is somehow related to the thyroid cell polarity. Immunofluorescence analyses of the localization of the two connexins in thyroid tissue sections revealed that connexin32 and connexin43 are located in different regions of the plasma membrane. We further analyzed the location of each of the two connexins with regard to that of the tight junction-associated protein, ZO1. Laser scanning confocal microscope observations of connexin32 or connexin43 and ZO1 double-immunolabelled thyroid cells, gave evidence for a separate localization of gap junctions made of each of these two connexins. Connexin32 gap junctions appeared as fluorescent spots scattered over the lateral membrane domain, while connexin43 gap junctions formed a meshed network superimposable with that of tight junctions in the subapical region of the cells. Western blot analyses of the distribution of connexins in thyroid plasma membrane subfractions obtained by ultracentrifugation on a sucrose gradient led to the identification of membrane sub-populations enriched in either connexin32 gap junctions or connexin43 gap junctions. Connexin32 gap junctions and connexin43 gap junctions were found to differ in their resistance to solubilization by N-lauroylsarcosine. Increasing concentrations of this detergent from 0.12% to 0.42% caused a progressive solubilization of connexin43 while connexin32 remained membrane-bound. These data demonstrate that connexin32 and connexin43, co-expressed by polarized thyroid cells, form separate gap junctions with distinct membrane location, gap junctions made up of connexin43 being precisely located within tight junctions. We report here, for the first time, a relationship between connexin distribution and cell polarity.
Inhibition of regulated catecholamine secretion from PC12 cells by the Ca2+/Calmodulin kinase II inhibitor KN-62
Authors: Schweitzer Erik S. , Sanderson Michael J. , Wasterlain C. G. 
Published: April 2021

ABSTRACT
When stimulated by the cholinergic agonist carbachol, PC12 cells rapidly secrete a large fraction of the intracellular catecholamines by exocytotic release from the large dense-core secretory vesicles in a Ca2+-dependent manner. To investigate whether Ca2+/calmodulin kinase II plays a role in the regulated secretion of catecholamines, we examined the effect of the specific Ca2+/calmodulin kinase II inhibitor KN-62 on the carbachol-induced release of norepinephrine from PC12 cells. Approximately 50% of the regulated release of norepinephrine, stimulated either by carbachol or direct depolarization, was inhibited by pretreatment with KN-62, while the remaining 50% was resistant to KN-62 and therefore independent of Ca2+/calmodulin kinase II. In contrast, H7, an inhibitor of protein kinase C, had no effect on any of the stimulated release. FURA 2 imaging experiments demonstrated that KN-62 does not act by blocking the stimulation-induced increase in intracellular [Ca2+]. The most likely model consistent with these data is that all the dense-core vesicles fuse with the plasma membrane in a Ca2+-dependent process, but that approximately 50% of the vesicles require an additional step that is dependent on the action of Ca2+/calmodulin kinase II. This step occurs between the influx of Ca2+ and the fusion of vesicle membranes with the plasma membrane, and may be analogous to the Ca2+/calmodulin kinase II phosphorylation of synapsin which mobilizes small, clear synaptic vesicles for exocytosis at the synapse.
Transcriptional activation of endothelial cell integrin αv by protein kinase C activator 12(S)-HETE
Authors: Tang Dean G. , Diglio Clement A. , Bazaz Rajesh , Honn Kenneth V. 
Published: April 2021

ABSTRACT
Previous work demonstrated that 12(S)-HETE [12(S)hydroxyeicosatetraenic acid], a lipoxygenase metabolite of arachidonic acid, stimulates the surface expression of integrin αvβ3 on mouse lung vascular endothelial cells (CD clone 3) in a post-transcriptional and protein kinase C (PKC)-dependent fashion. In this study we examined the effect of 12(S)-HETE on the expression of integrin receptors αvβ3 and α5β1 in a different clone of a mouse endothelial cell population derived from lung microvasculature (designated CD clone 4). The results indicated that 12(S)-HETE transcriptionally activates the gene expression of integrin αv as assessed by quantitative reverse transcription/polymerase chain reaction/Southern hybridization, RNase protection assay, solution hybridization, and northern blotting. The induction of αv mRNA occurred within 1 hour, peaked at ∼4 hours (2-to 4-fold increase), persisted for up to 16 hours, and thereafter gradually declined. The PKC activator phorbol 12-myristate 13-acetate (PMA) induced the αv mRNA, in a similar way. 12(S)-HETE treatment did not, in contrast, alter the mRNA levels of integrin subunit α5 or β1. The induction of αv mRNA appeared to be protein synthesis-independent, since cycloheximide did not alter the 12(S)-HETE effect. 12(S)-HETE also did not appear to alter the mRNA halflife of αv. On the other hand, 12(S)-HETE-induced increase in αv mRNA levels was PKC-dependent, since pretreatment of CD clone 4 cells with calphostin C significantly inhibited 12(S)-HETE-increased αv mRNA. Nuclear runoff experiments revealed that the increase in αv mRNA results from an enhanced gene transcription. Facilitated αv gene transcription resulted in an increased surface expression of αvβ3 protein, which resulted in an increased cell adhesion to vitronectin. The above observations, in conjunction with our previous experimental data, suggest that 12(S)-HETE may employ diverse mechanisms to stimulate the integrin αvβ3 expression in vascular endothelial cells, which could play important roles in tumor cell adhesion, angiogenesis, hemostasis, and many other vascular events.
Interactions of macromolecules with the mammalian cell surface
Authors: Wall Jonathan , Ayoub Fayad , O”Shea Paul 
Published: April 2021

ABSTRACT
The characterisation of fluoresceinphosphatidylethanol amine (FPE) as a real-time indicator of the electrostatic nature of the cell membrane surface is described. The con ditions appropriate for the labelling of cell membranes and the implementation of FPE as a tool to monitor the inter actions of various proteins and peptides with membranes are outlined. Some complications attributed to the ery throcyte glycocalyx are examined. In addition it is shown using neuraminidase as an example, that some types of enzyme-catalysed reactions on the cell surface may be monitored in real time. It is also shown that information concerning the binding of several proteins such as serum albumin and monoclonal antibodies are accessible with this technique. The albumin in particular is shown to exhibit a saturation of binding, the analysis of which indicates that the dissociation constant for erythrocytes was determined to be 8 µM and for lymphocytes to be almost 3 µM. On the basis of this comparison together with artificial membranes, the membrane protein components of the lym phocyte surface are implicated in the binding of albumin or the erythrocyte membrane proteins reduce the affinity of the cell surface for albumin.
Expression and glycosylation of the filamentous brush border glycocalyx (FBBG) during rabbit enterocyte differentiation along the crypt-villus axis
Authors: Maury J. , Bernadac A. , Rigal A. , Maroux S. 
Published: April 2021

ABSTRACT
The filamentous brush border glycocalyx forming the “enteric surface coat” of the intestinal epithelium is composed in rabbits of a 400 kDa mucin-type glycoprotein, which was purified using the 3A4 monoclonal antibody. This monoclonal antibody recognizes a filamentous brush border glycocalyx-specific glycosidic structure containing an O-acetylated sialic acid, which is absent from all the other glycoproteins in the epithelium, with the exception of certain goblet cell mucins. Here we establish that only 50% of the rabbits tested synthesized this glycosidic structure. Upon immunolabeling surface epithelia and sections of jejunum from these rabbits, the carbohydrate epitope rec ognized by the 3A4 mAb was found to be present on the fil amentous brush border glycocalyx of a variable number of enterocytes, which were patchily distributed over all the villi. This heterogeneous expression of 3A4 antigenicity, which was also observed in the crypts, suggests the existence of differences between the patterns of differen tiation of enterocytes, which results in the expression of different pools of glycosyltransferases and/or acetyl trans ferases. In mature enterocytes, the 3A4 determinants were present only on the filamentous brush border glycocalyx, which is anchored solely to the membrane microdomain at the tip of brush border microvilli. However, expression of 3A4 antigenicity begins in the median third of crypts, in enterocytes with a short, thin brush border devoid of apical filamentous brush border glycocalyx. Here the 3A4 epitopes were present over the whole brush border membrane. A few cells higher up, the hyperpolarized expression of filamentous brush border glycocalyx, i.e. its segregation at the tip of the microvilli, began and both the apical filamentous brush border glycocalyx and the whole brush border membrane were labeled with the 3A4 mAb. The labeling of the lateral membrane of microvilli gradually decreased and then disappeared during the migration of the cells to the upper part of the crypts. The 3A4 structure was also detected in some of the granules of some rare goblet cells in the vicinity of the most deeply located labeled enterocytes, whereas no labeled goblet cells were ever observed at higher cell locations.
Mitotic disassembly of the Golgi apparatus in vivo
Authors: Misteli Tom , Warren Graham 
Published: April 2021

ABSTRACT
Populations enriched in prophase cells were obtained either by using a cell line with a temperature-sensitive mutation in the mitotic kinase, p34cdc2, or by treating cells with olomoucine, an inhibitor of this kinase. Both methods resulted in efficient and reversible block of the cells at the G2/M boundary. After cells were released from the cell cycle block, the morphological changes to the Golgi apparatus were characterised using both quantitative con ventional electron microscopy and immuno-gold microscopy. The early mitotic phases were divided into six stages (G2 to pro-metaphase) based on the morphology of the nucleus. During prophase the cross-sectional length of Golgi stacks decreased prior to unstacking. At the same time, small vesicular profiles, typically 50-70 nm in diameter, accumulated in the vicinity of the stacks. The dis appearance of Golgi stacks was accompanied by the transient appearance of tubular networks. By the time cells entered prometaphase, the stacks had completely disas sembled and only clusters consisting of Golgi vesicles and short tubular elements were left. When cells were released from the G2/M boundary and pulsed briefly with [AlF4]- to prevent uncoating of transport vesicles, vesicular profiles with a morphology reminiscent of COP-coated vesicles appeared. These vesicular profiles were either associated with Golgi stacks or, at later stages, with clusters, but were formed at all stages of disassembly. Together these results provide further support for our model that continued budding of vesicles from the rims of Golgi cisternae is at least partly responsible for the disassembly of the Golgi apparatus.
Induction and regulation of connexin26 by glucagon in primary cultures of adult rat hepatocytes
Authors: Kojima Takashi , Mitaka Toshihiro , Shibata Yosaburo , Mochizuki Yohichi 
Published: April 2021

ABSTRACT
In the adult rat hepatocyte, the gap junction proteins consist of a major component, connexin32 (Cx32) and a minor component, connexin26 (Cx26). Although we recently reported our success in inducing and maintaining Cx32 in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor and 2% dimethyl sulfoxide, it was very difficult to induce Cx26 in the primary hepatocytes. In the present study, we found that the addition of 10−7 M glucagon into the culture medium could dramatically induce Cx26 mRNA and protein. Although the expression of Cx32 mRNA was also influenced by glucagon, the increase of the expression was small. Immunocytochemically, Cx26-positive spots were observed between most adjacent cells and were co-localized with the Cx32-positive spots. We also examined whether 0.5 mM dibutyl cyclic AMP could induce expression of Cx26 in the cells. The effect of dexamethasone on the expression of Cx26 mRNA compared to that of Cx32 mRNA was examined. For the induction and maintenance of Cx26 mRNA, more than 10−7 M dexamethasone was necessary in this culture. These results suggest that expression of Cx26 in hepatocytes may be regulated by the concentrations of glucagon and glucocorticoid hormones.
Protein translation components are colocalized in granules in oligodendrocytes
Authors: Barbarese Elisa , Koppel Dennis E. , Deutscher Murray P. , Smith Candra L. , Ainger Kevin , Morgan Frank , Carson John H. 
Published: April 2021

ABSTRACT
The intracellular distribution of various components of the protein translational machinery was visualized in mouse oligodendrocytes in culture using high resolution fluorescence in situ hybridization and immunofluorescence in conjunction with dual channel confocal laser scanning microscopy. Arginyl-tRNA synthetase, elongation factor 1a, ribosomal RNA, and myelin basic protein mRNA were all co-localized in granules in the processes, veins and membrane sheets of the cell. Colocalization was evaluated by dual channel cross correlation analysis to determine the correlation index (% colocalization) and correlation distance (granule radius), and by single granule ratiometric analysis to determine the distribution of the different components in individual granules. Most granules contained synthetase, elongation factor, ribosomal RNA and myelin basic protein mRNA. These results indicate that several different components of the protein synthetic machinery, including aminoacyl-tRNA synthetases, elongation factors, ribosomes and mRNAs, are colocalized in granules in oligodendrocytes. We propose that these granules are supramolecular complexes containing all of the necessary macromolecular components for protein translation and that they represent a heretofore undescribed subcellular organization of the protein synthetic machinery. This spatial organization may increase the efficiency of protein synthesis and may also provide a vehicle for transport and localization of specific mRNAs within the cell.
An anti-αv-integrin antibody that blocks integrin function inhibits the development of a human melanoma in nude mice
Authors: Mitjans Francesc , Sander Diane , Adán Jaime , Sutter Arne , Martinez Josep M. , Jäggle Carlota-S. , Moyano Josep M. , Kreysch Hans-Georg , Piulats Jaime , Goodman Simon L. 
Published: April 2021

ABSTRACT
A series of murine monoclonal antibodies were raised against purified human αvβ3 integrin and against M21 human melanoma cells. Five notable hybridomas were identified by ELISA on purified integrins, and the isolated antibodies bound the αv-chain. These antibodies, 17E6, 20A9, 23G5, 14D9.F8 and 10G2, recognised the extracellular domains of the integrin, and were shown to be reactive in FACS, immunoprecipitation, ELISA, and ELISA on fixed cells with M21, M21-L4, and UCLA-P3, but not with the αv-deficient M21-L or M21-L-IIb (M21-L transfected with GpIIb integrin).
One antibody, 17E6, strongly perturbed cell attachment mediated by αv integrins, reacting at least with αvβ3, αvβ5, and αvβ1, and strongly inhibiting cell attachment to αv-ligands vitronectin and fibronectin with an IC50 of ∼0.1 µg ml-1. Furthermore, 17E6 at this concentration could induce cell retraction from the substrate, while LM609 (anti-αvβ3) and control antibody 14E2 (anti-200 kDa melanoma surface protein) at 1,000-fold higher concentrations had minimal effects on cell morphology. The action of 17E6 was reversible and was not due to toxic effects: in vitro 17E6 at 0.1 mg ml-1 did not affect either cell proliferation or DNA synthesis.
In two nude-mouse tumour models, subcutaneous tumour development and a lung colonisation (‘experimental metastasis’) assay, injection of 17E6 strongly inhibited tumour development, while isotype-matched controls had no effect. There was no obvious mechanism of cell or of complement-mediated tumour cytotoxicity; the antibody did not mediate ADCC or AECDC, or complement fixation. The data strongly support previous studies which have indicated the importance of αv-integrins, and especially αvβ3, in the tumour progression of human melanoma.
Short- and long-term mechanisms of tau regulation in PC12 cells
Authors: Sadot Einat , Barg Jacob , Rasouly David , Lazarovici Phillip , Ginzburg Irith 
Published: April 2021

ABSTRACT
Induction by nerve growth factor of neurite outgrowth in PC12 cells is transcription-dependent and is associated with the accumulation of tau protein. It was recently shown that short-term treatment with staurosporine, a protein kinase alkaloid inhibitor, induced an elevation of tau protein levels and outgrowth of stable neurites. In this study, we analyzed the mechanism(s) by which nerve growth factor and staurosporine exert their effects on tau levels. We demonstrate that nerve growth factor affects tau mRNA stability, thus contributing to the observed increase in tau mRNA levels. On the other hand, tau mRNA levels were not affected by the treatment with staurosporine. We also demonstrate that the phosphorylation of tau protein was reduced after treatment of PC12 cells with nerve growth factor or staurosporine, as shown by immunoblot analysis using specific antibodies and alkaline phosphatase treatment. Thus, regulation of tau levels by nerve growth factor appears to be mediated by transcriptional, post-transcriptional and posttranslational steps, whereas the effect of staurosporine on tau levels may be attributed to its effect on the state of phosphorylation of the protein.
Protocadherins and diversity of the cadherin superfamily
Authors: Suzuki Shintaro T. 
Published: April 2021

ABSTRACT
Recent cadherin studies have revealed that many cadherins and cadherin-related proteins are expressed in various tissues of different multicellular organisms. These proteins are characterized by the multiple repeats of the cadherin motif in their extracellular domains. The members of the cadherin superfamily are divided into two groups: classical cadherin type and protocadherin type. The current cadherins appear to have evolved from a protocadherin type. Recent studies have proved the cell adhesion role of classical cadherins in embryogenesis. In contrast, the bio-logical role of protocadherins is elusive. Circumstantial evidence, however, suggests that protocadherins are involved in a variety of cell-cell interactions. Since protocadherins, and many other new cadherins as well, have unique properties, studies of these cadherins may provide insight into the structure and biological role of the cadherin superfamily.