bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of the integrin α6β4 in peripheral nerves: localization in schwann and perineural cells and different variants of the β4 subunit
Authors: Niessen C. M. , Cremona O. , Daams H. , Ferraresi S. , Sonnenberg A. , Marchisio P. C. 
Published: April 2021

ABSTRACT
Integrin α6β4 is expressed in human peripheral nerves, but not in the central nervous system. This integrin het-erodimer has previously been found in perineural fibrob-last-like cells and in Schwann cells (SCs), which both assemble a basement membrane but do not form hemidesmosomes. We show here that in SCs, which had formed a myelin sheath, α6β4 was enriched in the proximity of the nucleus, at Ranvier paranodal areas and at Schmitt-Lanterman clefts; α6β4 was also found at the grooved interface between small axons and non-myelinating SCs. Immunoprecipitation of human peripheral nerves, in com-bination with Western blotting showed that β4 is associated with the α6A subunit. Northern blot analysis of human peripheral nerves showed a single β4 transcript of 6 kb. Using the reverse transcriptase polymerase chain reaction, we detected two mRNA species, one for the most common (−70, −53) form of β4 and the other encoding the (+53) variant of β4. Cultured SCs were devoid of α6β4 but expressed α6β1, indicating that SCs lose β4 expression when contact with neurons is lost. Thus, resting SCs in contact with axons express α6A in combination with β4, irrespec-tive of myelin formation. We suggest that α6β4 expressed in SCs plays a role in peripheral neurogenesis.
Compartmentalization, processing and redistribution of the plasma membrane protein CE9 on rodent spermatozoa: Relationship of the annulus to domain boundaries in the plasma membrane of the tail
Authors: Cesario Mario M. , Bartles James R. 
Published: April 2021

ABSTRACT
Western blotting, immunofluorescence and immunogold electron microscopy were used to examine the compartmentalization, processing and redistribution of the integral plasma membrane protein CE9 on the spermatozoa of rats, mice and hamsters. In each species examined, spermatozoal CE9 was found to undergo endoproteolytic processing followed by a net redistribution from the posterior-tail domain into the anterior-tail domain of the plasma membrane during epididymal maturation. Compared to spermatozoa of the rat and mouse, those of the hamster were found to express a greater proportion of their CE9 within the anterior-tail plasma membrane domain at all stages of maturation. As a consequence, CE9 was judged to be a suitable marker for two different spermatozoal plasma membrane domains: the posterior-tail plasma membrane domain (spermatozoa from the testis and caput epididymidis of the rat and mouse) and the anterior-tail domain (spermatozoa from the cauda epididymidis of the hamster). Immunogold electron microscopy was used to pinpoint the positions of the boundaries of these CE9-con-taining plasma membrane domains at a high level of resolution. In each case, the position of the CE9 domain boundary was found to be strongly correlated with that of the subplasmalemmal electron-dense ring known as the annulus. The precise spatial relationship between the CE9 domain boundary and the annulus was, however, found to differ significantly among species and/or as a function of maturation.
The expression of proliferation-dependent antigens during the lifespan of normal and progeroid human fibroblasts in culture
Authors: Kill Ian R. , Faragher Richard G. A. , Lawrence Kay , Shall Sydney 
Published: April 2021

ABSTRACT
Normal human fibroblasts display a limited lifespan in culture, which is due to a steadily decreasing fraction of cells that are able to proliferate. Using antibodies that react with antigens present in proliferating cells only, in an indirect immunofluorescence assay, we have estimated the fraction of proliferating cells in cultures of normal human fibroblasts. Furthermore, we have estimated the rate of decline in the fraction of proliferating cells during the process of cellular ageing by application of the assay to normal human fibroblasts throughout their lifespan in culture. Werner’s Syndrome is an autosomal recessive disease in which individuals display symptoms of ageing prematurely. Werner’s Syndrome fibroblasts display a reduced lifespan in culture compared with normal human fibroblasts. Like normal human fibroblasts, the growth of Werner’s Syndrome fibroblasts is characterised by a decreasing fraction of cells reacting with the proliferation-associated antibodies throughout their lifespan in culture. However, the rate of loss of proliferating cells in Werner’s Syndrome fibroblasts during the process of cellular ageing is accelerated 5-to 6-fold compared with the rate deter-mined for normal human fibroblasts.
The regulation of chemotaxis and chemokinesis in Dictyostelium amoebae by temporal signals and spatial gradients of cyclic AMP
Authors: Vicker Michael G. 
Published: April 2021

ABSTRACT
The tactic and kinetic locomotion of Dictyostelium dis- coideum amoebae were examined in cyclic AMP (cAMP) spatial gradient and temporal signal fields. The distribu- tions of migrating cells were examined within 150 µm-thick micropore filters after incubation with different cAMP concentrations, [cAMP], applied in three ways across the fields: as positively or negatively developing gradients, generated either by increasing or decreasing the [cAMP] on one side of the filter, respectively, or as static, linear gradients after negative development. Chemotaxis was only induced by oriented, temporally increasing [cAMP]. Pulses propagated by molecular diffusion or mechanical flow were equally effective. Negatively developing cAMP gradients had no initial effect on cell accumulation. However, if the subsequent static spatial gradient was maintained by an infusion system, some gradients also induced cell accumu- lation, whose degree and direction depended on the gradient [cAMP]. The basis of this new effect was examined by tracking individual cells by computer-assisted videomi- croscopy during locomotion in different [cAMP]. Cells produced a triphasic [cAMP]-dependent response, with optimal cell motility induced by 10-30 nM. The results demonstrate that cell accumulation either up-field or down-field in spatial gradients is governed by the field locations of the attractant concentrations that induce the relative locomotory maxima and minima in the gradient field. Cells perceive the ambient [cAMP], but cannot read the spatial gradient orientation in static or yet steeper regions of developing gradients. Accumulation in static spatial gradients is a function of klino- and orthokinesis, but chemotaxis requires an oriented cAMP pulse or impulse. A mechanism of tactic signal perception is proposed in terms of the recently discovered intracellular oscillator, which determines cell shape and movement.
Characterisation of the paxillin-binding site and the C-terminal focal adhesion targeting sequence in vinculin
Authors: Wood C. K. , Turner C. E. , Jackson P. , Critchley D. R. 
Published: April 2021

ABSTRACT
Paxillin and vinculin are cytoskeletal proteins that co- localise to focal adhesions, specialised regions of the cell involved in attachment to the extracellular matrix. These two molecules form part of a complex of proteins that link the actin network to the plasma membrane. Paxillin has been shown to bind directly in vitro to the C-terminal region of vinculin (Turner et al. (1990). J. Cell Biol. 111, 1059-1068), which also contains a focal adhesion targeting sequence (Bendori et al. (1989). J. Cell Biol. 108, 2383- 2393). In the present study, we have used a series of vinculin deletion mutants to map more precisely the sites in vinculin responsible for paxillin binding and focal adhesion localisation. A glutathione-S-transferase fusion protein spanning vinculin residues 881-1000 was sufficient to support 125I-paxillin binding in a gel-blot assay while no detectable binding was observed to a fusion protein spanning residues 881-978. Transfection experiments using cDNAs encoding chick vinculin residues 398-1066 and 398- 1028 demonstrated that amino acids C-terminal to residue 1028 were not necessary for targeting to focal adhesions. In contrast, a vinculin polypeptide expressed from a cDNA encoding residues 398-1000 failed to localise to focal adhesions in stably transfected NIH3T3 cells. We have therefore identified a region of 50 amino acids (residues 979-1028) within the C-terminal region of vinculin that contains both the paxillin-binding site and the focal adhesion targeting sequence. This region is highly conserved in human and chicken vinculin and is likely to be important in regulation of the assembly of focal adhesions.
Rearrangement of mRNAs for costamere proteins during costamere development in cultured skeletal muscle from chicken
Authors: Morris E. J. , Fulton A. B. 
Published: April 2021

ABSTRACT
Mature skeletal myofibrils are surrounded by costameres, ribs of metavinculin, vinculin, intermediate filaments, and other proteins that connect the myofibril to the extracellular matrix. Costameres have recently been shown to be the sites at which the forces generated by the myofibril are transduced laterally into the extracellular matrix. We observed costameres developing in cultured skeletal muscles, grown in micromass culture from cells taken from embryonic chicken leg. We detected proteins by immunofluorescence and mRNA by in situ hybridization. Antibody and probe signals were imaged by laser scanning confocal microscopy. Antibody to vimentin protein is first detected in stripes in register with the Z line of the myofibril, at ∼day 12 after fusion; soon thereafter probe to vimentin mRNA is also detected in the same stripes. Optical sections indicate that vimentin mRNA and protein are very close, no more than 0.1 mm apart and possibly in immediate contact. Antibody to vimentin is detected in stripes only in cells that twitch spontaneously. Antibodies and probes to desmin and vinculin protein and mRNA are next detected in stripes of the same periodicity, at ∼day 17 after fusion. Vinculin protein (but not mRNA) is detected at focal contacts much earlier in development. Controls for bleed through of fluorescence, RNase H sensitivity, hybridization without probe, and binding to the myofibril all gave appropriate results. Probes to glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme, stained diffusely and did not associate with the myofibril. These results show that components of the costamere arrive at the structure in a defined sequence, and that mRNA organization is a conspicuous, precise and temporally controlled aspect of costamere development.
These results may have wider implications. In these cells, some mRNAs are positioned with submicrometer precision in space and differentially over time. Particular mRNAs differ in the time and place of such positioning. This implies both that cellular structures provide physical cues for such positioning and that mRNA contains information that interacts with such cues in a message-specific manner. If such precision in mRNA location is found in other somatic cells, it could have significant implications for the ways in which cells generate and maintain cellular structures.
Dictyostelium prespore-specific gene Dp87 encodes a sorus matrix protein
Authors: Nakao Hajime , Yamamoto Akitugu , Takeuchi Ikuo , Tasaka Masao 
Published: April 2021

ABSTRACT
In this paper we report on the characteristics of the product of a prespore-specific gene (Dp87) of Dictyostelium discoideum. Polyclonal antibody was made against a bacterially synthesized Dp87-encoded protein fragment. Using this antibody, the product was characterized by immunochemical and immunocytological methods. It was shown that the Dp87-encoded protein is a prespore-specific protein with a molecular mass of 83 kDa, which first appears at the standing slug stage and persists in mature fruiting bodies. Western blot studies revealed the presence of an additional 81 kDa protein prior to the appearance of the 83 kDa protein from the tipped aggregate to the standing slug stage, thus indicating the former to be a precursor protein. Immunocytochemical and immunoelectron microscopic studies showed that the protein is bound to ER at the early stages of development when only the 81 kDa protein is present. At the later stages when the 83 kDa protein predominates, however, it becomes localized in prespore-specific vacuoles (PSVs) and is associated with the inner fibrous material of PSVs, but not with the peripheral membranous material. This is in contrast to spore coat proteins, which are localized in PSVs from the beginning of their appearance and associated with both structures of PSVs. In mature fruiting bodies, most Dp87 protein is localized to the interspore space (matrix) of the sori, with some left on the surface of the stalk tube. Disruptants of the Dp87 gene were also produced. Although they contained neither 81 kDa nor 83 kDa protein, they showed no phenotypic defects as compared to the parental strain.
A 50 kDa protein present in conditioned medium of COLO-16 cells stimulates cell spreading and motility, and activates tyrosine phosphorylation of Neu/HER-2, in human SK-BR-3 mammary cancer cells
Authors: Corte Veerle De , Potter Christian De , Vandenberghe Dirk , Laerebeke Nicolas Van , Azam Mohamad , Roels Hendrik , Mareel Marc , Vandekerckhove Joël 
Published: April 2021

ABSTRACT
A factor present in conditioned medium of COLO-16 human cancer cells causes fast spreading, fast plasma membrane ruffling, cell shape change, net translocation, stimulation of chemotaxis and growth arrest in human SK-BR-3 mammary cancer cells. Based on the spreading effect, the factor was purified to homogeneity and migrated as a 50 kDa protein in SDS-polyacrylamide gel electrophoresis. Addition of the purified 50 kDa factor to the target cells in culture results in tyrosine phosphorylation of the p185erbB2 receptor concomitant with a fast redistribution and clustering of the receptor. The 50 kDa factor is also specifically retained by affinity chromatog-raphy on the immobilized extracellular domain of p185erbB2. Antibodies directed against this domain also inhibit the induction of motility. These data suggest that the 50 kDa factor is a putative ligand of p185erbB2 in SK-BR-3 cells. Biochemical and immunological evidence further indicate that this factor differs from p185erbB2 ligands described so far. Its activity could play a role in the pathogenesis of breast cancer.
The spatial localization of 18 S rRNA genes, in relation to the descent of the cells, in the root cortex of Petunia hybrida
Authors: Montijn M. B. , Houtsmuller A. B. , Nanninga J. L. Oud and N. 
Published: April 2021

ABSTRACT
The 3-D localization of transcription inactive 18 S rRNA genes was studied in interphase nuclei of Petunia hybrida root tip cells. To enable a cell type (i.e. cortex)-specific study in which also the orientation and descent of the cells could be taken into account, a method was developed to preserve the spatial organization of the root meristem. The ribosomal genes were detected by fluorescence in situ hybridization using a biotinylated cDNA probe. 3-D images of 81 nuclei, obtained by confocal scanning laser microscopy, were processed with newly developed computer software. 3-D nucleolar and nuclear dimensions, and the localization of the FISH-spots, were recorded interactively. We compared the absolute and relative position of the genes within and between files of cells of the cortex region of several roots, taking into account the genealogical relationship of the cells. Statistical analysis showed that both the relative and absolute positions of the inactive genes were random, also in more closely related cells within a file of cells. A ‘relict telophase orientation’ of the genes (i.e. the position of the genes in the daughter cells are mirror images of each other) could only be observed in the G0/1 phase of ‘true’ daughter cells; the orientation was not preserved throughout the next cell cycle.
Targeting of chimeric Gαi proteins to specific membrane domains
Authors: Almeida J. Bruno de , Holtzman Eliezer J. , Peters Philip , Ercolani Louis , Ausiello Dennis A. , Stow Jennifer L. 
Published: April 2021

ABSTRACT
Heterotrimeric guanine nucleotide-regulatory (G) proteins are associated with a variety of intracellular membranes and specific plasma membrane domains. In polarized epithelial LLC-PK1 cells we have shown previously that endogenous Gαi-2 is localized on the basolateral plasma membrane, whereas Gαi-3 is localized on Golgi membranes. The targeting of these highly homologous Gαi proteins to distinct membrane domains was studied by the transfection and expression of chimeric Gαi proteins in LLC-PK1 cells. Chimeric cDNAs were constructed from the cDNAs for Gαi-3 and Gαi-2 and introduced into a pMXX eukaryotic expression vector containing a mouse metallothionein-I promotor. Stably transfected cell lines were produced that expressed either Gαi-2/3 or Gαi-3/2 chimeric proteins. Chimeric and endogenous Gαi proteins were detected in cells using specific carboxy-terminal peptide antibodies. Immunofluorescence staining was used to localize en-dogenous and chimeric Gαi proteins in LLC-PK1 cells. The staining of chimeric proteins was detected as an increased intensity of staining on membranes containing endogenous Gαi proteins. Using confocal microscopy and image analysis we localized Gαi-2 to a specific sub-domain of the lateral membrane of polarized cells, the chimeric Gαi-3/2 protein was then shown to colocalize with endognenous Gαi-2 in the same lateral plasma membrane domain. The chimeric Gαi-2/3 protein colocalized with endogenous Gαi-3 on Golgi membranes in LLC-PK1 cells. These results show that chimeric Gαi proteins were targeted to the same membrane domains as endogenous Gαi proteins and the specificity of their membrane targeting was conferred by the carboxy-terminal end of the proteins. These data provide the first evidence for specific targeting information contained in the carboxy termini of Gαi proteins, which appears to be independent of amino-terminal membrane attachment sites in these proteins.
A three-dimensional reconstruction study of the rough ER-Golgi interface in serial thin sections of the pancreatic acinar cell of the rat
Authors: Sesso Antonio , Paulo de Faria Flávio , Iwamura Edna Sadayo Miazato , Corrêa Hélio 
Published: April 2021

ABSTRACT
Distinctive views of the tubulo-vesicular elements interposed between the endoplasmic reticulum (ER) and the Golgi apparatus were obtained in thin sections. The tubules that protrude from the transitional rough ER (tRER) are of dissimilar length. The numbers of tubules and of the nearby omega- and pear-shaped profiles decrease after fasting and are partially restored by refeeding. This formation is designated herein as the budding chamber of the tRER. Close to the budding chamber, clusters of 56 nm diameter vesicles are consistently observed. In some of the cells, convoluted tubules appear enmeshed with the presumptive transport vesicles of 56 nm diameter and with irregular, vesicular formations. Apparently structureless, electron-lucent ellipsoidal areas are found adjacent to these membranous elements. Serial and semi-serial sections show that the budding chamber, the sinuous tubules, the irregular vesicles, the structureless regions and the 56 nm vesicles fill tunnel-like spaces limited by the outermost Golgi cisterna (OGC) on one side and by the tRER on the other. Curved tubules appear to link the lumen of the OGC with that of smooth membranous occupants of these tunnel-like spaces. A presumptive luminal connection between these membranous occupants and the tubules of the budding chamber can also be seen. The predominant configuration of the OGC is that of a perforated, flat saccule. However, OGC regions exhibiting progressively lower densities of fenestrae, including smooth surfaced sectors eventually accumulating an intraluminal content are seen. Two such dilated, saccular portions of the OGC were analyzed through reconstruction of serial sections. Bundles of microtubules run closely apposed to the cis side of the OGC.
Molecular chaperones in pancreatic tissue: the presence of cpn10, cpn60 and hsp70 in distinct compartments along the secretory pathway of the acinar cells
Authors: Vélez-Granell Carlos S. , Arias Ariel E. , Torres-Ruíz José A. , Bendayan Moïse 
Published: April 2021

ABSTRACT
Three chaperones, the chaperonins cpn10 and cpn60, and the hsp70 protein, were revealed by immunochemistry and cytochemistry in pancreatic rat acinar cells. Western immunoblotting analysis of rat pancreas homogenates has shown that antibodies against cpn10, cpn60 and hsp70 protein recognize single protein bands of 25 kDa, 60 kDa and 70 kDa, respectively. Single bands for the cpn10 and cpn60 were also detected in pancreatic juice. Immunofluo-rescence studies on rat pancreatic tissue revealed a strong positive signal in the apical region of the acinar cells for cpn10 and cpn60, while an immunoreaction was detected at the juxtanuclear Golgi region with the anti-hsp70 antibody. Immunocytochemical gold labeling confirmed the presence of these three chaperones in distinct cell compartments of pancreatic acinar cells. Chaperonin 10 and cpn60 were located in the endoplasmic reticulum, Golgi apparatus, condensing vacuoles and secretory granules. Interestingly, the labeling for both cpn10 and cpn60 followed the increasing concentration gradient of secretory proteins along the RER-Golgi-granule secretory pathway. On the contrary, the labeling for hsp70 was mainly concentrated in the endoplasmic reticulum and the Golgi apparatus. In the latter, the hsp70 was found to be primary located in the trans-most cisternae and to colocalize with acid phosphatase in the trans-Golgi network. The three chaperones were also present in mitochondria. In view of the role played by the chaperones in the proper folding, sorting and aggregation of proteins, we postulate that hsp70 assists the adequate sorting and packaging of proteins from the ER to the trans-Golgi network while cpn10 and cpn60 play key roles in the proper packaging and aggregation of secretory proteins as well as, most probably, in the prevention of early enzyme activation in secretory granules.