bluuebunny/crossref_metadata_2025_split huggingface dataset:
Limpet Haemocytes I. Studies on Aggregation and Spike Formation
Authors: Davies P. S. , Partridge T. 
Published: April 2021

ABSTRACT
In this paper a promising new spontaneously aggregating system of amoebocytes from the blood of limpets is described. The system has the advantage of being readily available without cell culture and of being operational at room temperature.
The blood of the limpet Patella contains 2 types of haemocytes, amoebocytes and macrophages, the latter accounting for less than 1 % of the cell population. The concentration of cells in suspension varies with the body temperature of the animal, from about 1 ×108 cells/ml at 5 °C to 9 × 108 cells/ml at 25 °C.
Following blood withdrawal the amoebocytes are approximately spherical in shape with superficial convoluted lamellae, but over a period of 5 min develop stout marginal spikes. This development is temperature dependent and independent of divalent cations in the medium.
Most interestingly, and apparently associated with the stimulus of blood withdrawal, the cells become spontaneously adhesive and commence an extremely rapid aggregation. With a shaking speed of 700 strokes/min the half time of aggregation varies from 15 s at an initial cell density of 4·7 × 106 cells/ml to 60 s at a density of 1·4 × 106 cells/ml. The aggregates so formed are very resistant to mechanical dispersion and tight junctions have been observed between aggregated cells in electron micrographs. Complete aggregation requires the presence of divalent cations and another unidentified plasma component. Aggregation will occur at 0°C but the process is slower and less complete than at 20 °C. It is suggested that aggregation involves the triggering or activation of a pre-synthesized adhesive system and that the adhesiveness per unit area increases with increase in temperature.
Embryoglycans regulate FGF-2-mediated mesoderm induction in the rabbit embryo
Authors: Dvořák P. , Flechon J.-E. , Thompson E. M. , Horák V. , Adenot P. , Renard J. P. 
Published: April 2021

ABSTRACT
Several peptide growth factors, including members of the fibroblast growth factor (FGF) superfamily, are potential inducers of mesoderm in vertebrates. Receptor binding of basic FGF (FGF-2) is promoted by cell surface or extracellular matrix proteoglycans. The substantial biosynthesis of proteoglycans by embryonic cells (called embryoglycans) and their potential role as ligands for growth factor receptors led us to examine the role of embryoglycans that carry the developmentally regulated oligosaccharide epitope TEC 1, in the binding of FGF-2 to cultured rabbit inner cell masses (ICMs).
Culture of isolated ICMs in the presence of FGF-2 gave rise to well delimited colonies with migrating cells at the periphery. In these cells, TEC 1 staining shifts from a punctate pattern over the entire membrane, to an apical, finely granular distribution with some internalization. This shift occurs after 96 hours in culture.
Here we show that: (1) migrating cells are mesoderm-like in phenotype; (2) antibodies against TEC 1 blocked FGF-2 mediated differentiation in vitro; (3) antibodies against TEC 1 selectively blocked binding of FGF-2 to ectodermal receptors and, vice versa, the binding of TEC 1-specific antibodies to ectodermal cells can be competed by excess FGF-2; (4) the same switch in TEC 1 staining patterns was observed in vivo, between the day 7 and the day 9 rabbit embryo.
These data suggest the involvement of defined species of embryonic cell surface epitopes in the regulation of FGF-2 receptor binding. Moreover, this proposed binding activity is temporally restricted to ectodermal cells and disappears early during differentiation. Thus, the apical TEC 1 redistribution can be considered as the earliest indicator of mesoderm formation.
Protein phosphatases maintain the organization and structural interactions of hepatic keratin intermediate filaments
Authors: Toivola Diana M. , Goldman Robert D. , Garrod David R. , Eriksson John E. 
Published: April 2021

ABSTRACT
The importance of protein phosphatases in the maintenance of cytoskeletal structure is supported by the serious liver injury caused by microcystin-LR, a hepatotoxic inhibitor of type-1 and type-2A serine/threonine protein phosphatases. We used the microcystin-LR-induced cell injury as a model to study the roles of protein dephosphorylation in maintaining cytoskeletal structure and cellular interactions in primary rat hepatocyte cultures. Confocal microscopy revealed that the first visible effect of micro-cystin-LR is disruption of desmoplakin organization at the cell surface, indicating dissociation of desmosomes. This effect is followed by a dramatic reorganization of both the intermediate filament (keratins 8 and 18) and microfilament networks, resulting in a merged structure in which the intermediate filaments are organized around a condensed actin core. Keratin 8, keratin 18 and desmo-plakin I/II are the major cytoskeleton-associated targets for microcystin-LR-induced phosphorylation. Hyperphosphorylation of keratin 8 and 18 is accompanied by an increased keratin solubility, which correlates with the observed mor-phological effects. Phosphopeptide mapping shows that four specific tryptic phosphopeptides are highly phosphorylated predominantly in the soluble pool of keratin 18, whereas keratin 8 shows no indications of such assembly state-specific sites. Phosphopeptide maps of keratins phosphorylated in vivo and in vitro indicate that Ca2+/calmod-ulin-dependent kinase may be involved in regulating the serine-specific phosphorylation of both keratin 8 and keratin 18, while cAMP-dependent protein kinase does not seem to play a major role in this context. Taken together, our results show that the interactions between keratin intermediate filaments and desmosomes as well as the assembly states of their main constituent proteins, are directly regulated by serine/threonine kinase/phosphatase equilibria.
ERD2 proteins mediate ER retention of the HNEL signal of LRP’s receptor-associated protein (RAP)
Authors: Bu Guojun , Rennke Stephanie , Geuze Hans J. 
Published: April 2021

ABSTRACT
The 39 kDa receptor-associated protein (RAP) is a receptor antagonist that interacts with several members of the low density lipoprotein (LDL) receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors. Our recent studies have demonstrated that RAP is an endoplasmic reticulum (ER) resident protein and an intracellular chaperone for the LDL receptor-related protein (LRP). The HNEL sequence at the carboxyl terminus of RAP represents a novel ER retention signal that shares homology with the well-characterized KDEL signal. In the present study, using immunoelectron microscopy we demonstrate that cells stably transfected with human growth hormone (GH) tagged with either KDEL (GH+KDEL) or HNEL (GH+HNEL) signals exhibit ER and cis-Golgi localization typical of ER-retained proteins. Overexpression of not only GH+HNEL but also GH+KDEL cDNA in transfected cells results in saturation of ER retention receptors and secretion of endogenous RAP indicating that the two signals interact with the same ER retention receptor(s). The role of RAP in the maturation of LRP is further supported by the observation that functional LRP is reduced about 60% as a result of decreased intracellular RAP. Pulse-chase labeling and immunolocalization studies of ERD2.1 and ERD2.2 proteins in transfected cells demonstrate a long half-life and Golgi localization for both receptors. Finally, overexpression of either ERD2.1 or ERD2.2 proteins significantly increases the capacity of cells to retain both KDEL and HNEL-contain-ing proteins. Taken together, our results thus demonstrate that ERD2 proteins are capable of retaining the novel ER retention signal associated with RAP.
Translocation of a UV-damaged DNA binding protein into a tight association with chromatin after treatment of mammalian cells with UV light
Authors: Rapić Otrin Vesna , McLenigan Mary , Takao Masashi , Levine Arthur S. , Protić Miroslava 
Published: April 2021

ABSTRACT
A UV-damaged DNA binding protein (UV-DDB) is the major source of UV-damaged DNA binding activity in mammalian cell extracts. This activity is defective in at least some xeroderma pigmentosum group E (XP-E) patients; microinjection of the UV-DDB protein into their fibroblasts corrects nucleotide excision repair (NER). In an in vitro reconstituted NER system, small amounts of UV-DDB stimulate repair synthesis a few fold. After exposure to UV, mammalian cells show an early dose-dependent inhibition of the extractable UV-DDB activity; this inhibition may reflect a tight association of the binding protein with UV-damaged genomic DNA. To investigate the dynamics and location of UV-DDB with respect to damaged chromatin in vivo, we utilized nuclear fractionation and specific antibodies and detected translocation of the p127 component of UV-DDB from a loose to a tight association with chromatinized DNA immediately after UV treatment.
A similar redistribution was found for other NER proteins, i.e. XPA, RP-A and PCNA, suggesting their tighter association with genomic DNA after UV. These studies revealed a specific protein-protein interaction between UV-DDB/p127 and RP-A that appears to enhance binding of both proteins to UV-damaged DNA in vitro, providing evidence for the involvement of UV-DDB in the damage-recognition step of NER. Moreover, the kinetics of the reappearance of extractable UV-DDB activity after UV treatment of human cells with differing repair capacities positively correlate with the cell’s capacity to repair 6-4 pyrimidine dimers (6-4 PD) in the whole genome, a result consistent with an in vivo role for UV-DDB in recognizing this type of UV lesion.
Cytomechanics of neurite outgrowth from chick brain neurons
Authors: Chada Sandeep , Lamoureux Phillip , Buxbaum Robert E. , Heidemann Steven R. 
Published: April 2021

ABSTRACT
Mechanical tension is a direct and immediate stimulus for neurite initiation and elongation from peripheral neurons. We report here that the relationship between tension and neurite outgrowth is equally initimate for embryonic chick forebrain neurons. Culture of forebrain neurons was unusually simple and reliable, and some of these cells undergo early events of axonal-dendritic polarity. Neurite outgrowth can be initiated de novo by experimental application of tension to the cell margin of forebrain neurons placed into culture 8-12 hours earlier, prior to spontaneous neurite outgrowth. Experimentally induced neurite elongation from these neurons shows the same robust linear relationship between elongation rate and magnitude of applied tension as peripheral neurons, i.e. both show a fluid-like growth response to tension. Although forebrain and sensory neurons manifest a similar distribution of growth sensitivity to tension (growth rate/unit tension), chick forebrain neurons initiated and elongated neurites at substantially lower net tensions than peripheral neurons. This is because, unlike peripheral neurons, there is no minimum threshold tension required for elongation in forebrain neurons; all positive tensions stimulate neurite outgrowth. Consistent with this observation, chick forebrain neurons showed weak retractile behavior in response to slackening compared to sensory neurons. Neurites that were slackened showed only transient elastic behavior and never actively produced tension, as do chick sensory neurons after slackening. We conclude that tension is an important regulator of both peripheral and central neuronal growth, but that elastic behavior is much weaker for forebrain neurons than peripheral neurons from the same developing organism. These data have significance for the understanding of the morphogenetic events of brain development.
Molecular cloning, over-expression, developmental regulation and immunolocalization of fragminP, a gelsolin-related actin-binding protein from Physarum polycephalum plasmodia
Authors: T’Jampens Davy , Meerschaert Kris , Constantin Bruno , Bailey Juliet , Cook Lynnette J. , Corte Veerle De , Mol Hans De , Goethals Mark , Damme José Van , Vandekerckhove Joël , Gettemans Jan 
Published: April 2021

ABSTRACT
FragminP is a Ca2+-dependent actin-binding and micro-filament regulatory protein of the gelsolin family. We screened a Physarum polycephalum cDNA library with polyclonal fragminP antibodies and isolated a cDNA clone of 1,104 bp encoding 368 amino acids of fragminP, revealing two consensus phosphatidylinositol 4,5 bisphosphate-binding motifs in the central part of the protein. The first methionine is modified by an acetyl group, and three amino acids were missing from the protein coded for by the cDNA clone. Full-length recombinant fragminP was generated by PCR, purified after over-expression from Escherichia coli and displayed identical properties to native Physarum fragminP. Northern blot analysis against RNA, isolated from cultures at various stages of development, indicated that fragminP is absent from amoebae and that expression is initiated at an early stage during apogamic development, in a similar way to that observed for the profilin genes. In situ immunolocalization of fragminP in Physarum microplasmodia revealed that the protein is localized predominantly at the plasma membrane, suggesting a role in the regulation of the subcortical actin meshwork. Our data indicate that we have isolated the plasmodium-specific fragminP cDNA (frgP) and suggest that, in each of its two vegetative cell types, P. polycephalum uses a different fragmin isoform that performs different functions.
Mesenchyme-mediated effects of retinoic acid during rat intestinal development
Authors: Plateroti Michelina , Freund Jean-Noël , Leberquier Cathy , Kedinger Michèle 
Published: April 2021

ABSTRACT
In previous experiments we showed that intestinal development was dependent upon epithelial-mesenchymal cell interactions. The aim of this study was to investigate the possible role of retinoic acid (RA), a morphogenetic and differentiating agent, on the gut epithelial-mesenchymal unit. For this purpose we first analyzed the effects of a physiological dose of RA on 14-day fetal rat intestine using short-term organ culture experiments, or long-term grafts under the skin of nude mice. In these conditions, RA accelerated villus outgrowth and epithelial cell differentiation as assessed by the onset of lactase expression, and it also stimulated muscle and crypt formation. In order to analyze potential effects of RA mediated by mesenchymal cells, we isolated and characterized gut mucosa mesenchyme-derived cell cultures (mesenchyme-derived intestinal cell lines, MIC). These cells were shown to express mRNAs for retinoid binding proteins similar to those expressed in situ in the intestinal mesenchyme. MIC cells co-cultured with 14-day intestinal endoderms promoted endodermal cell adhesion and growth, and the addition of exogeneous RA enhanced epithelial cell polarization and differentiation assessed by cytokeratin and lactase immunostaining. Such a differentiating effect of RA was not observed on endo-dermal cells when cultured without a mesenchymal feeder layer or maintained in conditioned medium from RA-treated MIC cells. In the co-cultures, immunostaining of laminin and collagen IV with polyclonal antibodies, as well as α1 and β1 laminin chains mRNAs (analyzed by RT-PCR) increased concurrently with the RA-enhanced differentiation of epithelial cells. It is worth noting that this stimulation by RA was also obvious on the mesenchymal cells cultured alone. These results show that RA plays a role in intestinal morphogenesis and differentiation. In addition, they indicate that RA acts on the mesenchymal cell phenotype and suggest that RA may modify the mes-enchymal-epithelial cell interactions during intestinal development.
Pathogen-induced programmed cell death in tobacco
Authors: Mittler Ron , Simon Lee , Lam Eric 
Published: April 2021

ABSTRACT
Sacrificing an infected cell or cells in order to prevent systemic spread of a pathogen appears to be a conserved strategy in both plants and animals. We studied some of the morphological and biochemical events that accompany programmed cell death during the hypersensitive response of tobacco plants infected with tobacco mosaic virus. Certain aspects of this cell death process appeared to be similar to those that take place during apoptosis in animal cells. These included condensation and vacuolization of the cytoplasm and cleavage of nuclear DNA to 50 kb fragments. In contrast, internucleosomal fragmentation, condensation of chromatin at the nuclear periphery and apoptotic bodies were not observed in tobacco plants during tobacco mosaic virus-induced hypersensitive response. A unique aspect of programmed cell death during the hypersensitive response of tobacco to tobacco mosaic virus involved an increase in the amount of monomeric chloroplast DNA. Morphological changes to the chloroplast and cytosol of tobacco cells and increase in monomeric chloroplast DNA occurred prior to gross changes in nuclear morphology and significant chromatin cleavage. Our findings suggest that certain aspects of programmed cell death may have been conserved during the evolution of plants and animals.
Receptor-mediated endocytosis of urokinase-type plasminogen activator is regulated by cAMP-dependent protein kinase
Authors: Goretzki Lothar , Mueller Barbara M. 
Published: April 2021

ABSTRACT
Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/α2-macroglobulin (α2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RIα of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RIα protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RIα antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated α2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.
Glycosaminoglycans modulate fibronectin matrix assembly and are essential for matrix incorporation of tenascin-C
Authors: Chung Chang Y. , Erickson Harold P. 
Published: April 2021

ABSTRACT
We have investigated the role of glycosaminoglycans in fibronectin matrix assembly and the incorporation of tenascin-C into matrix fibrils. Chinese hamster ovary cell mutants with a total block in heparan and chondroitin sulfate production failed to assemble a fibronectin matrix, and incorporated no tenascin-C. Another mutant with reduced heparan sulfate produced a normal fibronectin matrix but failed to incorporate tenascin-C. Excess soluble glycosaminoglycans inhibited the binding of tenascin-C to purified fibronectin in ELISA, and completely blocked incorporation into matrix fibrils. Treating cultured cells with xyloside, which interferes with glycosaminoglycan attachment to proteoglycans, also completely blocked their ability to incorporate tenascin-C into matrix fibrils. We conclude that proteoglycans bound to fibronectin fibrils play a major role in binding tenascin-C to these fibrils. We examined more closely the large heparan sulfate proteoglycan, perlecan, and found that it co-localizes with tenascin-C and fibronectin in the matrix. The perlecan binding site in tenascin-C was mapped to the fibronectin type III domains 3-5, but this binding was strongly enhanced for the small splice variant, which is the major form incorporated into the matrix. Apparently when the alternative splice segment is inserted after domain 5 it inhibits perlecan binding. Thus heparan sulfate glycosaminoglycans, and perlecan in particular, may play a role in incorporation of the small splice variant of tenascin-C into fibronectin matrix fibrils.
Molecular chaperones and the cytoskeleton
Authors: Liang P. , MacRae T. H. 
Published: April 2021

ABSTRACT
Heat shock proteins, first observed because they are preferentially synthesized by organisms exposed to heat or other physiological stress, are also synthesized constitutively. These proteins are divided into several families, namely, HSP100, 90, 70, 60 (chaperonin), and the small heat shock/α-crystallin proteins. They enjoy a wide phylogenetic distribution and are important because they function as molecular chaperones, able to mediate many cellular processes through an influence on higher order protein structure. For example, molecular chaperones assist in the transport of proteins into mitochondria and chloroplasts, as well as influencing clathrin lattice dynamics, viral replication and transcriptional activation. Under conditions of stress, some molecular chaperones prevent denaturation of proteins while others may dissociate protein aggregates, refolding monomers derived therefrom or directing their proteolytic destruction. We present in this review an analysis of the emerging literature on the relationship between molecular chaperones and the cytoskeleton, a collection of polymeric structures consisting of microtubules, microfilaments and intermediate filaments. A recent development in this field is identification of the TCP-1 complex as the eukaryotic cytoplasmic chaperonin which directs folding of cytoskeletal proteins such as α/β/γ-tubulin, actin and centractin. Moreover, the TCP-1 complex is a centrosomal component, apparently involved in the nucleation of microtubules. Other molecular chaperones recognize one or more cytoskeletal elements and in most cases they modulate the assembly of and/or provide protection for their constituent proteins. For example, HSP70 protects the centrosome and perhaps intermediate filaments during heat shock, and like HSP90, it binds to microtubules. Small heat shock proteins interact with microfilaments and intermediate filaments, affect their polymerization and guard them from heat shock by a phosphorylation-dependent mechanism. We conclude that molecular chaperones have different but cooperative roles in the formation and function of the eukaryotic cell cytoskeleton.