bluuebunny/crossref_metadata_2025_split huggingface dataset:
A mutation in a highly conserved region in brush-border sucrase-isomaltase and lysosomal α-glucosidase results in Golgi retention
Authors: Moolenaar Catharina E. C. , Ouwendijk Joke , Wittpoth Michael , Wisselaar Heleen A. , Hauri Hans-Peter , Ginsel Leo A. , Naim Hassan Y. , Fransen Jack A. M. 
Published: April 2021

ABSTRACT
A point mutation in the cDNA of human intestinal sucrase-isomaltase has been recently identified in phenotype II of congenital sucrase-isomaltase deficiency. The mutation results in a substitution of glutamine by proline at position 1098 (Q1098P) in the sucrase subunit. Expression of this mutant sucrase-isomaltase cDNA in COS-1 cells results in an accumulation of sucrase-isomaltase in the ER, intermediate compartment and the cis-Golgi cisternae similar to the accumulation in phenotype II intestinal cells. An interesting feature of the Q1098P substitution is its location in a region of the sucrase subunit that shares striking similarities with the isomaltase subunit and other functionally related enzymes, such as human lysosomal acid α-glucosi-dase and Schwanniomyces occidentals glucoamylase. We speculated that the QrP substitution in these highly conserved regions may result in a comparable accumula tion. Here we examined this hypothesis using lysosomal α-glucosidase as a reporter gene. Mutagenesis of the glutamine residue at position 244 in the homologous region of α-glucosidase to proline results in a protein that is neither transported to the lysosomes nor secreted extracel-lularly but accumulates in the ER, intermediate compartment and cis-Golgi as a mannose-rich polypeptide similar to mutant sucrase-isomaltase in phenotype II.
We propose that the Q1098P and Q244P mutations (in sucrase-isomaltase and α-glucosidase, respectively) generate structural alterations that are recognized by a control mechanism, operating beyond the ER in the intermediate compartment or cis-Golgi.
VE-cadherin antibody accelerates neutrophil recruitment in vivo
Authors: Gotsch Ursula , Borges Eric , Bosse Roland , Böggemeyer Elena , Simon Markus , Mossmann Horst , Vestweber Dietmar 
Published: April 2021

ABSTRACT
Neutrophils enter sites of inflammation by crossing the endothelial lining of the blood vessel wall. VE-cadherin is an endothelial specific, homophilic adhesion molecule located at the lateral cell surface. We have generated a monoclonal antibody against mouse VE-cadherin which inhibits electrical resistance of endothelial cell monolayers in vitro as well as aggregation of VE-cadherin transfected cells. In vivo, this antibody was found to increase vascular permeability and to accelerate the entry of neutrophils into chemically inflamed mouse peritoneum. Thus, VE-cadherin is essential for the integrity of the endothelial barrier in vivo. Our data suggest that opening of VE-cadherin mediated endothelial cell contacts may be a relevant step during neutrophil extravasation.
Sorting of MHC class II molecules and the associated invariant chain (li) in polarized MDCK cells
Authors: Simonsen Anne , Stang Espen , Bremnes Bjørn , Røe Målfrid , Prydz Kristian , Bakke Oddmund 
Published: April 2021

ABSTRACT
Epithelial cells have been found to express MHC class II molecules in vivo and are able to perform class II-restricted antigen presentation. The precise intracellular localization of these molecules in epithelial cells has been a matter of debate. We have analyzed the polarized targeting of human MHC class II molecules and the associated invariant chain (Ii) in stably transfected MDCK cells. The class II molecules are located at the basolateral surface and in intracellular vesicles, both when expressed alone or together with Ii. Ii is located in basolateral endosomes and can internalize through the basolateral plasma membrane domain. We show that the cytoplasmic tail of Ii contains information for basolateral targeting as it is sufficient to redirect the apical protein neuraminidase (NA) to the baso lateral surface. We find that the two leucine-based motifs (LI and ML) in the cytoplasmic tail of Ii are individually sufficient for endosomal sorting and basolateral targeting of Ii in MDCK cells. In addition, basolateral sorting information is located within the 10 membrane-proximal residues of the Ii cytoplasmic tail. As several different signals mediate basolateral sorting of the class II/Ii complex, a polarized distribution of these molecules may be an essential feature of antigen presentation in epithelial cells.
Ca2+-dependent interaction of S100A2 with muscle and nonmuscle tropomyosins
Authors: Gimona Mario , Lando Zeev , Dolginov Yakov , Vandekerckhove Joel , Kobayashi Ryuji , Sobieszek Apolinary , Helfman David M. 
Published: April 2021

ABSTRACT
Zero-length chemical crosslinking with 1-ethyl-3-[3-(dimethyl amino)propyl]carbodiimide (EDC) indicated an association of the Ca2+-binding protein S100A2 with tropomyosin (TM) in vitro. The mobility of the crosslinked product on SDS-PAGE gels indicated the formation of a 1:1 complex between S100A2 and TM and the interaction was Ca2+ dependent. Monoclonal antibodies were raised against S100A2 and used to determine its cellular localization in the porcine epithelial cell line LLC PK1. It was found that the localization of S100A2 depended on the differentiation state of the cells, being absent from actin stress fibers in sparsely seeded cultures, but present in the actin-containing microvilli characteristic of differentiated cells. Immunoprecipitations of [35S] methionine-labeled extracts using S100A2 as well as TM-specific antibodies failed to co-precipitate TM and S100A2, indicating a transient association between these two molecules in solution. Affinity chromatography of cell extracts on immobilized recombinant TMs, however, confirmed the Ca2+-dependent interaction between S100A2 and both muscle TMs as well as with high and low molecular mass nonmuscle TMs, suggesting that the binding site resides in one of the conserved regions of TM. Our data demonstrate the possible interaction of S100A2 with TM that is not bound to the microfilaments and indicate a differentiation-related function for S100A2 in LLC PK1 cells. The possible functional implications of this interaction are discussed.
The developmentally regulated Drosophila embryonic nuclear lamina protein ‘Young Arrest’ (fs(1)Ya) is capable of associating with chromatin
Authors: Lopez Jacqueline M. , Wolfner Mariana F. 
Published: April 2021

ABSTRACT
The Drosophila fs(1)Ya protein (YA) is an essential component of the early embryonic nuclear lamina. Mutant zygotes lacking functional YA arrest in the first division cycles following fertilization, hence having a ‘Young Arrest’ of their development. The nuclear lamina is thought to act as the structural backbone for the nucleus and to provide anchoring sites for interphase chromosomes. Here, we demonstrate in vitro that YA is not required for the de novo formation of nuclear structures. Since YA’s sequence predicts potential DNA binding motifs, this protein may instead function to connect the lamina and chromosomes, and thus aid in organizing the nucleus. We ectopically expressed YA in polytene cells and demonstrated its association with polytene chromosomes, preferentially at interbands. Furthermore, our in vitro studies indicate that embryonic YA protein is capable of associating with decondensed chromatin. These observations suggest that YA may be required for the interaction between chromatin and the nuclear envelope during early embryogenesis.
Acid modulation of HT29 cell growth and differentiation An in vitro model for Barrett’s esophagus
Authors: Fitzgerald Rebecca C. , Omary M. Bishr , Triadafilopoulos George 
Published: April 2021

ABSTRACT
Cell proliferation and differentiation are influenced by environmental factors, including the extracellular pH. We recently showed, using an ex vivo organ culture system of human mucosal Barrett’s esophageal biopsies, that acid has a highly variable effect on cell proliferation and differentiation depending on the pattern of acid exposure. Study of the mechanisms underlying these dynamic effects of acid on this premalignant intestinal-like epithelium is hampered by lack of an immortalized cell line. We therefore investigated the effect of acid exposure on the human colonic carcinoma cell line HT29, chosen because of its intestinal cell derivation and its ability to differentiate in vitro. HT29 cells exposed to pH 5 medium either continuously (up to 3 weeks), or as a short (1 hour) pulse, were compared with cells cultured at pH 7.4. Villin expression was induced only by long term acid exposure, and correlated with the development of differentiated polarized cells that contain a brush border and microvillus inclusions. Chronic acid exposure arrested cell proliferation, whereas a 1 hour acid-pulse enhanced cell proliferation, as determined by [3H]thymidine incorporation assays and proliferating cell nuclear antigen expression. Serum starvation attenuated the hyperproliferative effect of an acid-pulse. In addition, the doubling time of at least the first cell cycle after an acidpulse was shortened. The Na/H exchanger is likely to play a role since the hyperproliferative acid-induced response was blocked by amiloride; and the activity of the exchanger was increased at acidic pH as determined by 22Na uptake. These results support a role for extracellular pH on cell proliferation and differentiation of HT29 cells. Furthermore, these findings parallel the dynamic effects of acid on Barrett’s esophagus, and suggest that HT29 cells could serve as an in vitro model for studying the mechanism of acid modulation in Barrett’s esophagus.
Retargeting of cytosolic proteins to the plasma membrane by the Lck protein tyrosine kinase dual acylation motif
Authors: Zlatkine P. , Mehul B. , Magee A. I. 
Published: April 2021

ABSTRACT
Several members of the Src family of protein tyrosine kinases have a N-terminal dual acylation motif which specifies their myristoylation and S-acylation. These lipid modifications are necessary for correct intracellular localisation to the plasma membrane and to detergent-resistant glycolipid-enriched membrane domains (GEMs). Using chimaeras of the Lck dual acylation motif with two normally cytosolic proteins (chloramphenicol acetyl transferase and galectin-3), we show here that this motif is sufficient to encode correct lipid modification and to target these chimaeras to the plasma membrane, as demonstrated by subcellular fractionation and confocal immunofluorescence microscopy of transiently transfected COS cells. In addition, the chimaeras are resistant to extraction with cold non-ionic detergent, indicating targeting to GEM subdomains in the plasma membrane. The dual acylation motif has potential for targeting proteins to specific plasma membrane subdomains involved in signalling.
Glucose deprivation-induced cytotoxicity in drug resistant human breast carcinoma MCF-7/ADR cells: role of c-myc and bcl-2 in apoptotic cell death
Authors: Lee Yong J. , Galoforo Sandra S. , Berns Christine M. , Tong William P. , Kim Hyeong Reh C. , Corry Peter M. 
Published: April 2021

ABSTRACT
We investigated the effect of glucose deprivation treatment on clonogenicity in multidrug-resistant human breast carcinoma MCF-7/ADR cells. Survival of MCF-7/ADR cells decreased exponentially up to 8 hours of incubation in the glucose-free medium. The surviving fraction of these cells for 8 hours of glucose-deprivation treatment was 1.5×10−3. Photomicrographs and gel electrophoresis data suggest that glucose deprivation-induced cell death is associated with apoptosis. Data from western and northern blots showed an induction of c-myc gene expression during treatment with glucose-free medium in MCF-7/ADR cells. MCF-7/ADR cells transfected with c-myc antisense oligodeoxynucleotides became resistant to glucose deprivation-induced apoptosis. Overexpression of bcl-2 gene protected MCF-7/ADR cells from this apoptotic cell death. Taken together, these results indicate that c-myc expression is a necessary component of glucose-free medium induced apoptosis and bcl-2 prevents apoptotic death induced by c-myc.
Oncostatin M induces basic fibroblast growth factor expression in endothelial cells and promotes endothelial cell proliferation, migration and spindle morphology
Authors: Wijelath Errol S. , Carlsen Brian , Cole Toby , Chen Jing , Kothari Sudesh , Hammond William P. 
Published: April 2021

ABSTRACT
Oncostatin M (OSM), a pleiotropic cytokine originally isolated from supernatants of the U937 histiocytic lymphoma cell line, has been shown to have regulatory effects on a wide variety of cultured and tumor cells. We investigated the effects of OSM on basic fibroblast growth factor (bFGF) gene expression in bovine arterial endothelial (BAE) cells. Levels of bFGF mRNA transcripts were low in uninduced BAE cells, were maximal at 8 hours of exposure to OSM, and returned to control levels by 24 hours. Induction of bFGF mRNA transcripts by OSM was dose-dependent. Nuclear transcriptional run-on analysis demonstrated that exposure of BAE cells to OSM stimulated bFGF gene transcription. OSM treatment of BAE cells enhanced the synthesis of bFGF protein as determined by ELISA assays. Immunocytochemistry studies demonstrated the presence of low levels of bFGF protein within the cytoplasm in uninduced cells. After stimulation for 8 hours with OSM there was significant staining for bFGF in the cytoplasm. However, 24 hours after exposure to OSM, bFGF antigen was located only within the nuclei. Western blot analysis demonstrated that OSM stimulated predominantly the synthesis of a 22 kDa form of bFGF. In addition, OSM stimulated endothelial cell proliferation and migration as well as acquisition of a spindle shape. Phosphorothioate antisense oligonucleotide directed against bFGF inhibited OSM induced BAE cell proliferation and spindle shape formation but had only a minimal effect on migration. The levels of the 22 kDa form of bFGF were reduced by antisense treatment indicating that OSM induced proliferation and morphology change is likely to be regulated by intracellular bFGF. Our studies suggest that OSM released at sites of vascular injury could stimulate angiogenesis by inducing bFGF synthesis, endothelial cell proliferation and migration.
A Drosophila Tpr protein homolog is localized both in the extrachromosomal channel network and to nuclear pore complexes
Authors: Zimowska Grażyna , Aris John P. , Paddy Michael R. 
Published: April 2021

ABSTRACT
Here we report structural, molecular, and biochemical characterizations of Bx34, a Drosophila melanogaster nuclear coiled-coil protein which is localized to extrachromosomal and extranucleolar spaces in the nuclear interior and which is homologous to the mammalian nuclear pore complex protein Tpr. In the nuclear interior, Bx34 is excluded from chromosomes and the nucleolus and generally localizes to regions between these structures and the nuclear periphery. This distribution matches the ‘extrachromosomal channel network’ described previously. In the nuclear periphery, Bx34 localizes on or near nuclear pore complexes. Biochemically, Bx34 isolates exclusively with the nuclear matrix fraction. The Bx34 cDNA sequence predicts a large protein (262 kDa) with two distinct structural domains. The Bx34 N-terminal 70% (180 kDa) is predicted to form an extended region of coiled-coil, while the C-terminal 30% (82 kDa) is predicted to be unstructured and acidic. Bx34 shows moderate sequence identity over its entire length to the mammalian nuclear pore complex protein ‘Tpr’ (28% amino acid identity and 50% similarity). Furthermore, several of the sequence motifs and biochemical similarities between Bx34 and Tpr are sufficiently striking that it is likely that Bx34 and Tpr are functionally related. The Bx34 gene exists in a single copy in region 48C of chromosome 2R. The localization of coiledcoil Bx34 to both the nuclear interior and nuclear pore complexes and its sequence similarity to a known nuclear pore complex protein leads to speculations about a role for Bx34 in nucleo-cytoplasmic transport which we can test using molecular genetic approaches.
The transcriptional basis of chromosome pairing
Authors: Cook P. R. 
Published: April 2021

ABSTRACT
Pairing between homologous chromosomes is essential for successful meiosis; generally only paired homologs recombine and segregate correctly into haploid germ cells. Homologs also pair in some somatic cells (e.g. in diploid and polytene cells of Drosophila). How homologs find their partners is a mystery. First, I review some explanations of how they might do so; most involve base-pairing (i.e. DNA-DNA) interactions. Then I discuss the remarkable fact that chromosomes only pair when they are transcriptionally active. Finally, I present a general model for pairing based upon the DNA-protein interactions involved in transcription. Each chromosome in the haploid set has a unique array of transcription units strung along its length. Therefore, each chromatin fibre will be folded into a unique array of loops associated with clusters of polymerases and transcription factors; only homologs share similar arrays. As these loops and clusters, or transcription factories, move continually, they make and break contact with others. Correct pairing would be nucleated when a promoter in a loop tethered to one factory binds to a homologous polymerizing site in another factory, before transcription stabilizes the association. This increases the chances that adjacent promoters will bind to their homologs, so that chromosomes eventually become zipped together with their partners. Pairing is then the inevitable consequence of transcription of partially-condensed chromosomes.
Lateral mobility of plasma membrane lipids in bull spermatozoa: heterogeneity between surface domains and rigidification following cell death
Authors: Ladha Shab , James Peter S. , Clark David C. , Howes Elizabeth A. , Jones Roy 
Published: April 2021

ABSTRACT
Compartmentalization of surface membrane antigens into discrete regions or domains is a characteristic feature of differentiated cells. In mammalian spermatozoa at least 5 surface domains are known, implying the presence of barriers or boundaries within the plasma membrane. Using the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusibility of fluorescent lipid analogues 1,1’-dihexadecyl-3,3,3’3’-tetramethylindo-carbocyanine (DiIC16) and 5-(N-octa-decanoyl) aminofluorescein (ODAF), we have investigated lipid topology and dynamics in the plasma membrane of ejaculated bull spermatozoa. Contrary to reports in the literature, we have found that DiIC16 stains only dead or damaged spermatozoa whereas ODAF intercalates into the plasma membrane of both live and dead cells, each type showing a distinctive staining pattern. FRAP analysis with ODAF revealed that diffusion coefficients on live spermatozoa are significantly faster on the acrosome and postacrosome (29.3×10−9 cm2/second) than on the midpiece and principal piece (11.8× 10−9 cm2/second). Recovery (R) is &gt;90% in all domains. ODAF diffusion also shows regionalized temperature-sensitivity with a 4-fold increase over the sperm head and a 1.8-fold increase on the tail between 20°C and 37°C. Remarkably, dead or permeabilized spermatozoa rapidly develop a large immobile phase (R&lt;25%) over the whole plasma membrane. This rigidification is temperature insensitive and irreversible suggesting major changes in the physical state of membrane lipids. It is concluded that lipid diffusion in the plasma membrane of live bull spermatozoa is rapid and varies significantly between surface domains. Following permeabilization or cell death, however, a large immobile phase develops indicating substantial changes in membrane lipid disposition.
Keratin intermediate filament dynamics in cell heterokaryons reveals diverse behaviour of different keratins
Authors: Paramio Jesús M. , Casanova M. Llanos , Alonso Angel , Jorcano José L. 
Published: April 2021

ABSTRACT
To study the dynamics of keratin intermediate filaments, we fused two different types of epithelial cells (PtK2 and BMGE+H) and studied how the keratins from the parental cells recombine and copolymerize to form the heterokaryon cytoskeleton. The behaviour of the keratins during this process was followed by immunofluorescence using specific antibodies. After fusion, the parental cytoskeletons undergo a depolymerization process most apparent in the region adjacent to the fusion area. The depolymerized subunits spread throughout the heterokaryon and copolymerize into a new hybrid cytoskeleton. The complete process is very rapid, occurring in 3-4 hours, thus demonstrating the highly dynamic nature of the keratin cytoskeleton. Although newly synthesised subunits contribute to the formation of the hybrid cytoskeleton, the process takes place with similar kinetics in the absence of protein synthesis, showing the dynamic nature of the keratins from pre-existing cytoskeletons. During this process, specific keratins behave differently. Keratins K8, K18, K5 and K10 are mobilised from the parental cytoskeletons and reassemble rapidly into the hybrid cytoskeleton (3-6 hours), whereas K14 requires a substantially longer period (9-24 hours). Thus, different keratins, even when they form part of the same heterodimeric/tetrameric complexes, as is the case for K5 and K14, exhibit different dynamics. This suggests that individual polypeptides or homopolymeric complexes rather than exclusively heterodimeric/ tetrameric subunits, as is currently thought, can also take part in keratin intermediate filament assembly and dynamics. Biochemical analysis performed in the absence of protein synthesis revealed greater amounts of K5 than of K14 in the soluble pool of BMGE+H cells. Crosslinking and immunoprecipitation experiments indicated an excess of monomeric K5, as well as of K5/K14 heterodimers and K5 homodimers in the soluble pool. These results are in agreement with the different dynamic behaviour of these keratins observed in immunofluorescence. On the contrary, the phosphorylation levels of K5 and K14 are similar in both the soluble pool and the polymerized fraction, suggesting that phosphorylation does not play an important role in the different dynamics displayed by these two proteins. In summary, our results demonstrate that, following fusion, the keratin intermediate filament network reshapes rather rapidly and that keratins are highly dynamic proteins, although this mobility depends on each particular polypeptide.