bluuebunny/crossref_metadata_2025_split huggingface dataset:
Visualization and quantification of glycolipid polarity dynamics in the plasma membrane of the mammalian spermatozoon
Authors: Gadella B. M. , Gadella T. W. J. , Colenbrander B. , Golde L. M. G. van , Lopes-Cardozo M. 
Published: April 2021

ABSTRACT
Seminolipid (sulphogalactosylalkylacylglycerol), the glycolipid that is specific for mammalian germ cells, is located exclusively in the outer leaflet of the sperm plasma membrane. In this study the lateral distribution of semi-nolipid on sperm heads has been investigated by indirect immunofluorescence labelling and detection with digital imaging fluorescence microscopy. In freshly ejaculated sperm cells this glycolipid was present primarily at the apical ridge subdomain of the plasma membrane of the sperm head. After binding the sperm cells to zona-coated coverslips seminolipid migrated, in 40 minutes, from the apical ridge to the equatorial subdomain of the plasma membrane. A similar redistribution of seminolipid was observed during capacitation of sperm cells in vitro induced by Ca2+ or bovine serum albumin. Comparable migration of seminolipid was also found after prolonged storage of ejaculated sperm cells, albeit at a much slower rate. Addition of arylsulphatase A, an enzyme present in seminal plasma that desulphates seminolipid, significantly enhanced the migration of seminolipid during storage of sperm cells. Its breakdown product desulphoseminolipid (galactosylalkylacylglycerol) appeared highly specifically at the equatorial segment. The measured fluorescence intensity over the sperm head surface correlated linearly with the spatial probe distribution as was checked by fluorescence lifetime imaging microscopy. This paper demonstrates and quantifies for the first time the polarity of semi-nolipid on the surface of the sperm cell and the dynamic alterations that occur in this polarity during post-ejaculatory events.
NSP-encoded reticulons, neuroendocrine proteins of a novel gene family associated with membranes of the endoplasmic reticulum
Authors: Velde Helgi J. K. van de , Roebroek Anton J. M. , Senden Nicole H. M. , Ramaekers Frans C. S. , Ven Wim J. M. Van de 
Published: April 2021

ABSTRACT
The novel NSP gene was previously shown to encode, among a variety of neuroendocrine cell types, two 3′-over-lapping transcripts, a 3.4 kb one for NSP-A (776 amino acids) and a 1.8 kb one for NSP-C (208 amino acids). The deduced proteins, which were predicted to possess distinct amino-terminal regions, appeared to exhibit some archi-tectural resemblance to known neuroendocrine proteins. In this paper the biochemical characterization and subcellu-lar localization of the two proteins is addressed. In vitro translation of NSP-A and -C RNA produced proteins of about 135 and 23 kDa, respectively. Proteins of similar molecular mass were also detected in immunoprecipitation and western blot analyses of neural and endocrine cells using specific anti-NSP-A or -C antisera; some heterogen-eity of NSP-A was observed. NSP-A, but not NSP-C, appeared to be highly phosphorylated and preferentially on serine residues. In immunocytochemical studies, we demonstrated that NSP-A and -C are associated with the endoplasmic reticulum; NSP-A was found to co-localize with SERCA2b, a membrane-associated Ca2+-ATPase of the endoplasmic reticulum. In Purkinje cells, we found NSP-immunostaining in the perikaryon, the extensive dendritic tree and the axon, also suggesting association with the smooth endoplasmic reticulum. Biochemical studies of NSP-A provided evidence that NSP-A is strongly associated with microsomal membranes and analysis of deletion mutants of NSP-A revealed that the hydrophobic carboxy-terminal portion of the protein, which is also present in NSP-C, is critical for membrane binding. Through database searches, finally, we found two different NSP-related sequences, one in a sequenced region of human chromosome 19, and the second in a human, pan-creatic islet-derived partial cDNA, suggesting that the NSP gene is the prototype of a larger gene family. The results of our studies seem to indicate that the NSP-encoded proteins are novel, membrane-anchored components of the endo-plasmic reticulum for which we propose the name reticulons.
Association of kinesin with the Golgi apparatus in rat hepatocytes
Authors: Marks David L. , Larkin Janet M. , McNiven Mark A. 
Published: April 2021

ABSTRACT
The Golgi apparatus is a dynamic membranous structure, which has been observed to alter its location and morphology during the cell cycle and after microtubule disruption. These dynamics are believed to be supported by a close structural interaction of the Golgi with the microtubule cytoskeleton and associated motor enzymes. One micro-tubule-dependent motor enzyme, kinesin, has been implicated in Golgi movement and function although direct evidence supporting this interaction is lacking. In this study, we utilized two well-characterized kinesin antibodies in conjunction with subcellular fractionation techniques, immunoblot analysis and immunofluorescence microscopy to conduct a detailed study on the association of kinesin with the Golgi and other membranous organelles in a polarized epithelial cell, the primary rat hepatocyte. We found that kinesin represents ∼0.3% of total protein in rat liver homogenates, with ∼30% membrane-associated and the remainder in the cytosol. Among membrane fractions, kinesin was concentrated markedly in Golgi-enriched fractions, which were prepared using two independent techniques. Kinesin was also abundant in fractions enriched in transcytotic carriers and secretory vesicles, with lower levels detected on fractions enriched in endosomes, endoplasmic reticulum, lysosomes and mito-chondria. Immunofluorescence microscopy showed that kinesin is concentrated on Golgi-like structures in both primary cultured hepatocytes and rat hepatocyte-derived clone 9 cells. Double-label immunofluorescence demonstrated that kinesin staining colocalizes with the Golgi marker, α-mannosidase II, in both cell types. These results provide compelling evidence showing that kinesin is associated with the Golgi complex in cells and implicate this motor enzyme in Golgi structure, function and dynamics.
Formation of high molecular mass DNA fragments is a marker of apoptosis in the human leukaemic cell line, U937
Authors: Bicknell Gareth R. , Snowden Roger T. , Cohen Gerald M. 
Published: April 2021

ABSTRACT
Inhibitors of macromolecular synthesis and topoisomerases induce apoptosis in the human leukaemic cell line, U937. In this study, U937 cells were treated with the RNA synthesis inhibitor, actinomycin D (1 μM), the protein synthesis inhibitors, emetine (1 μM) and cycloheximide (100 μM), the topoisomerase II inhibitor, teniposide (5 μM), or the topoisomerase I inhibitor, camptothecin (1 μM). Apoptotic cell death was assessed both by flow cytometry and agarose gel electrophoresis, and was correlated to the appearance of large (20 to ≥580 kilobase pairs) DNA fragments, as assessed by field inversion gel electrophoresis. In all cases, the appearance of DNA fragments of 20-50 kilobase pairs accompanied the appearance of an apoptotic population and of internucleosomal cleavage. However, teniposide additionally induced a marked increase in fragmentation to ≥580 kilobase pairs. The co-treatment of cells with zinc (1 mM) inhibited the formation of all large DNA fragments, internucleosomal cleavage and the appearance of an apoptotic population.
We conclude that the generation of large DNA fragments is characteristic of apoptosis induced by various stimuli in U937, as has been found previously in rat thymocytes. However, unlike what occurs in rat thymocytes, zinc treatment does not dissociate the formation of large fragments from conventional markers of apoptosis.
Inhibition of apical but not basolateral endocytosis of ricin and folate in Caco-2 cells by cytochalasin D
Authors: Jackman Mark R. , Shurety Wenda , Ellis Juliet A. , Luzio J. Paul 
Published: April 2021

ABSTRACT
Apical and basolateral endocytic pathways in polarised Caco-2 cells were investigated by following the uptake, recycling and transcytosis of the galactose-binding protein toxin ricin, as a membrane marker. Differences in the extent and kinetics of lectin uptake, recycling and transcy-tosis were observed at the apical and basolateral domains and altered with the age of the cell monolayer. Treatment of polarised Caco-2 cells with cytochalasin D showed a domain-specific, concentration-dependent inhibition of apical endocytosis of ricin. Inhibition of apical endocytosis by cytochalasin D was not due to a gross change in brush border morphology, although actin stress fibres within the cell body were disrupted. It is not clear whether inhibition of apical endocytosis in polarized epithelial cells by cytochalasin D is caused simply by disruption of a mechanochemical motor involving microvillar actin filaments. The cytochalasin D effect was also observed when measuring uptake of folate, suggesting apical domain-specific inhibition of caveolar, as well as clathrin-mediated, endocytic routes.
Utrophin actin binding domain: analysis of actin binding and cellular targeting
Authors: Winder S. J. , Hemmings L. , Maciver S. K. , Bolton S. J. , Tinsley J. M. , Davies K. E. , Critchley D. R. , Kendrick-Jones J. 
Published: April 2021

ABSTRACT
Utrophin, or dystrophin-related protein, is an autosomal homologue of dystrophin. The protein is apparently ubiq-uitously expressed and in muscle tissues the expression is developmentally regulated. Since utrophin has a similar domain structure to dystrophin it has been suggested that it could substitute for dystrophin in dystrophic muscle. Like dystrophin, utrophin has been shown to be associated with a membrane-bound glycoprotein complex. Here we demonstrate that expressed regions of the predicted actin binding domain in the NH2 terminus of utrophin are able to bind to F-actin in vitro, but do not interact with G-actin. The utrophin actin binding domain was also able to associate with actin-containing structures, stress fibres and focal contacts, when microinjected into chick embryo fibroblasts. The expressed NH2-terminal 261 amino acid domain of utrophin has an affinity for skeletal F-actin (Kd 19±2.8 μM), midway between that of the corresponding domains of α-actinin (Kd 4 μM) and dystrophin (Kd 44 μM). Moreover, this utrophin domain binds to non-muscle actin with a ∼4-fold higher affinity than to skeletal muscle actin. These data (together with those of Matsumura et al. (1992)Nature, 360, 588-591) demonstrate for the first time that utrophin is capable of performing a functionally equivalent role to that of dystrophin. The NH2 terminus of utrophin binds to actin and the COOH terminus binds to the membrane associated glycoprotein complex, thus in non-muscle and developing muscle utrophin performs the same predicted ‘spacer’ or ‘shock absorber’ role as dystrophin in mature muscle tissues. These data suggest that utrophin could replace dystrophin functionally in dystrophic muscle.
In vitro differentiation of embryonic stem cells into glial cells and functional neurons
Authors: Fraichard A. , Chassande O. , Bilbaut G. , Dehay C. , Savatier P. , Samarut J. 
Published: April 2021

ABSTRACT
Mouse embryonic stem cells were induced to differentiate in culture with retinoic acid. Putative precursors of neurons and glial cells (nestin-positive cells) were clearly identified as early as three days after the onset of differentiation. At day 6, neuron-like cells could be clearly identified, either as isolated cells or as cellular networks. Some of these cells were positive for astrocyteor oligodendrocyte-specific antigens (GFAP or O4 antigens, respectively). Other cells were positive for neuron-specific antigens (cytoskeleton proteins MAP2, MAP5 and NF200, as well as synaptophysin). Some neuronal-like cells were also positive for acetylcholinesterase activity or glutamic acid decarboxylase expression, indicating that ES cells could differentiate into GABAergic and possibly cholinergic neurons. Electrophysiological analyses performed in voltage clamp conditions showed that cell membranes contained voltagedependent channels. Overshooting action potentials could be triggered by current injection. Taken together, these data provide evidence that embryonic stem cells can differentiate first into neuron-glia progenitors, and later into glial cells and functional neurons, in vitro. This technique provides an unique system to study early steps of neuronal differentiation in vitro.
Does cytosolic free Ca2+ signal neutrophil chemotaxis in response to formylated chemotactic peptide?
Authors: Laffafian Iraj , Hallett M. B. 
Published: April 2021

ABSTRACT
Cytosolic free Ca2+ concentration was measured and imaged in human neutrophils moving towards a source of formylated peptide in a micropipette held close to the cells. Under these conditions, neutrophils changed shape and displayed chemotaxis without significant or persistent global or localised elevations in cytosolic free Ca2+. A rearto-front persistent Ca2+ gradient of less than 0.5 nM/μm was present in the migrating neutrophils, until they reached the zone of higher peptide concentration, when an abrupt rise in cytosolic free Ca2+ concentration was triggered and chemotaxis stopped. Small localised rises in cytosolic free Ca2+, which were occasionally observed during neutrophil manoeuvring, were attributed to the effect of local deformation of the neutrophil membrane, since deformation of the membrane with a blunt micropipette caused similar Ca2+ changes. These data suggest that neutrophil chemotaxis towards a source of formylated peptide occurs without significant changes in Ca2+ signalling.
Targeted disruption of the Dictyostelium myosin essential light chain gene produces cells defective in cytokinesis and morphogenesis
Authors: Chen Tung-Ling L. , Kowalczyk Patricia A. , Ho Guyu , Chisholm Rex L. 
Published: April 2021

ABSTRACT
We have previously demonstrated that the myosin essential light chain (ELC) is required for myosin function in a Dictyostelium cell line, 7-11, in which the expression of ELC was inhibited by antisense RNA overexpression. We have now disrupted the gene encoding the ELC (mlcE) in Dictyostelium by gene targeting. The mlcE−mutants provide a clean genetic background for phenotypic analysis and biochemical characterization by removing complications arising from the residual ELC present in 7-11 cells, as well as the possibility of mutations due to insertion of the antisense construct at multiple sites in the genome. The mlcE− mutants, when grown in suspension, exhibited the typical multinucleate phenotype observed in both myosin heavy chain mutants and 7-11 cells. This phenotype was rescued by introducing a construct that expressed the wildtype Dictyostelium ELC cDNA. Myosin purified from the mlcE− cells exhibited significant calcium ATPase activity, but the actin-activated ATPase activity was greatly reduced. The results obtained from the mlcE− mutants strengthen our previous conclusion based on the antisense cell line 7-11 that ELC is critical for myosin function. The proper localization of myosin in mlcE− cells suggests that its phenotypic defects primarily arise from defective contractile function of myosin rather than its mislocalization.
The enzymatic defect of myosin in mlcE− cells also suggests a possible mechanism for the observed chemotactic defect of mlcE− cells. We have shown that while mlcE− cells were able to respond to chemoattractant with proper directionality, their rate of movement was reduced. During chemotaxis, proper directionality toward chemoattractant may depend primarily on proper localization of myosin, while efficient motility requires contractile function. In addition, we have analyzed the morphogenetic events during the development of mlcE− cells using lacZ reporter constructs expressed from cell type specific promoters. By analyzing the morphogenetic patterns of the two major cell types arising during Dictyostelium development, prespore and prestalk cells, we have shown that the localization of prespore cells is more susceptible to the loss of ELC than prestalk cells, although localization of both cell types is abnormal when developed in chimeras formed by mixing equal numbers of wild-type and mutant cells. These results suggest that the morphogenetic events during Dictyostelium development have different requirements for myosin function.
Peripherin assembles into homopolymers in SW13 cells
Authors: Cui Cunqi , Stambrook Peter J. , Parysek Linda M. 
Published: April 2021

ABSTRACT
The properties of full-length and mutant peripherins were studied in intermediate filament-less SW13 cells to define regions of peripherin that are essential for initiation of filament assembly. A full-length rat peripherin gene transfected into SW13 cells resulted in filament formation, consistent with the close structural relationship of peripherin to other type III intermediate filament proteins that readily form homopolymers. Translation of full-length rat peripherin is initiated predominantly at the second of two inframe AUGs. Deletions within the amino terminus of wildtype peripherin abolished its ability to form filaments in SW13 cells. In contrast, deletion of the entire carboxylterminal tail of peripherin did not affect its ability to form filamentous arrays in transfected SW13 cells. These results indicate that, of the intermediate filament proteins that are expressed in mature neurons, only peripherin and αinternexin are capable of making homopolymer intermediate filaments. In addition, mutations of the carboxyl tail of peripherin generally do not interfere with filament network formation.
Identification of an hsp70-related protein associated with the centrosome from dinoflagellates to human cells
Authors: Perret Eric , Moudjou Mohammed , Geraud Marie Line , Derancourt Jean , Soyer-Gobillard Marie-Odile , Bornens Michel 
Published: April 2021

ABSTRACT
The monoclonal antibody CTR210 raised against isolated human centrosomes strongly decorates the centrosome and more weakly a domain congruent with the Golgi apparatus in several animal cells (HeLa, 3T3, CHO, PtK2). Both decorations resist Triton extraction in conditions which totally extract the Golgi apparatus, as judged by galactosyltransferase decoration. A 67 kDa centrosomal antigen can be demonstrated in human cells with this antibody. CTR210 also decorates the centrosome or associated structures in several systems, including unicellular eukaryotes such as dinoflagellates or ciliates. A 72 kDa antigen has been identified and purified from the dinoflagellate C. cohnii and its NH2-terminal sequence partially established. It shows a close homology with HSP70 proteins. The possibility that the 72 kDa antigen belongs to this chaperone family was further supported using a mAb reacting, in most species, with HSP70. A polyclonal antibody raised against the 72 kDa antigen from C. cohnii decorates the centrosome in human cells and reacts with the CTR210 centrosomal 67 kDa antigen. These results suggest that specific chaperone proteins are associated with the centrosome in eukaryotic cells. The centrosomal chaperones could participate in the microtubule nucleation reaction or in the process of centrosome assembly.
Tissue-specific and efficient expression of the human simple epithelial keratin 8 gene in transgenic mice
Authors: Casanova Llanos , Bravo Ana , Were Felipe , Ramírez Angel , Jorcano José J. , Vidal Miguel 
Published: April 2021

ABSTRACT
Keratin 8 is a type II intermediate filament protein found in simple epithelia. We have introduced a 12 kb DNA fragment of the human K8 locus into the germ line of mice. The transgene, containing 1.1 kb of 5′ flanking sequences, 7.7 kb corresponding to the body of the gene and 3.2 kb of 3′ flanking sequences, was expressed in all six lines obtained. Immunolocalization and RNA analysis of adult tissues showed that the tissue-specific expression pattern of the transgene was almost indistinguishable from that of the endogenous gene. This pattern was found in organs containing single epithelial cell types, such as trachea, lung, stomach, intestine, liver, kidney, thymus and glands. The highest expressing line, however, also produced human K8 in tissues such as stratified epithelia, where it formed part of the pre-existing keratin cytoskeleton of basal cells. Steady state levels of human K8 RNA were proportional to the copy number of the transgene, but transgene expression was less efficient, per gene copy, than that of the endogenous gene. When in the 12 kb DNA fragment the exons and introns of the gene were replaced by the Escherichia coli lacZ gene, the resulting construct showed no expression in transgenic mice. This suggests that 5′ and 3′ flanking sequences, in the absence of intragenic sequences, are not sufficient for K8 expression and that important control elements are located in the body of the K8 gene.
the role of α4β1 integrin in cell motility and fibronectin matrix assembly
Authors: Wu Chuanyue , Fields Andrew J. , Kapteijn Barbara A. E. , McDonald John A. 
Published: April 2021

ABSTRACT
The α4β1 integrin has been suggested to play important roles in embryogenesis and pathogenesis of many diseases which involve both cell adhesion and cell migration. Previous studies using anti-α4β1 antibodies and fibronectin (Fn) fragments have suggested that α4β1 integrins may be involved in cell motility on Fn and vascular cell adhesion molecule-1 (VCAM-1). However, the cells used in these studies also express other Fn integrin receptors including α5β1 integrin, which is known to function in cell motility on Fn. To test whether α4β1 integrins mediate cell motility on Fn and VCAM-1 in the absence of α5β1 integrin, we expressed human α4 integrin in a Chinese hamster ovary (CHO) cell line that is deficient in α5β1 integrin (CHO B2). The parental α5 deficient CHO B2 cells were unable to adhere, spread or migrate on Fn, nor could they assemble a fibrillar Fn matrix. Expression of α4β1 integrin in the CHO B2 cells enabled the cells to adhere, spread and migrate on Fn and on VCAM-1 but not to assemble a fibrillar Fn matrix. The cellular processes mediated by the interaction of α4β1 with Fn or VCAM-1 were inhibited by the CS1 peptide derived from the major α4β1 binding site on Fn. These findings demonstrate that α4β1 integrins not only function as cell adhesion receptors but also as cell motility receptors for Fn and VCAM-1 independent of α5β1. Moreover, they reveal important functional differences between Fn binding integrins. The α4-positive, α5-negative CHO cells described in this report will be useful tools in studying the mechanism of molecular signalling during integrin mediated cellular processes.