bluuebunny/crossref_metadata_2025_split huggingface dataset:
Endocytosis of folate-protein conjugates: ultrastructural localization in kb cells
Authors: Turek John J. , Low Philip S. 
Published: April 2021

ABSTRACT
It has been demonstrated that proteins covalently conjugated to folic acid may be taken up by cells via endo-cytosis after binding to a folate binding protein (FBP) in the cell membrane. The proteins taken up in this manner remain catalytically active and they may modify physiological processes occurring in the cytosol. Confocal fluorescence microscopy of KB cells incubated with FITC-bovine serum albumin-folic acid conjugates showed that after uptake, the conjugates resided in large vesicular structures. The purpose of the present study was to determine the subcellular localization of protein-folic acid conjugates in KB cells using folic acid-bovine serum albumin-colloidal gold (F-BSA-CG) as a tracer. F-BSA-CG conjugates were taken up via uncoated pits or caveolae, and resided primarily in multivesicular bodies (MVBs) and other tubular endosomes at early time points (15-60 min). At later time points (6 hours), conjugates were still contained in MVBs but some were also found in secondary lysosomes or free in the cytoplasm. Coincubation of KB cells with transferrin-colloidal gold (TF-CG) and F-BSA-CG resulted in colocalization of TF-CG and F-BSA-CG within endosomal elements at times later than 15 minutes, indicating that the caveolae-mediated F-BSA-CG endocytic pathway converged with a pathway utilized by clathrin-coated pits.
Immunological characterization of lamins in the nuclear matrix of onion cells
Authors: Mínguez A. , Díaz de la Espina S. Moreno 
Published: April 2021

ABSTRACT
We have used polyclonal and monoclonal antibodies against different lamins from vertebrates, and the IFA antibody recognizing all kinds of intermediate filament proteins, to investigate the lamins of the nuclear matrix of Allium cepa meristematic root cells. All the antibodies react in the onion nuclear matrix with bands in the range of 60-65 kDa, which are enriched in the nuclear matrix after urea extraction, and do not crossreact with other antibodies recognizing intermediate filaments in plants (AFB, anti-vimentin and MAC 322), ruling out crossreaction with contaminating intermediate filaments of cytoplasmic bundles. In 2-D blots the chicken antilamin serum reacts with one spot at 65 kDa and pI 6.8 and the anti B-type lamin antibodies with another one at 64 kDa and pI 5.75. Both crossreact with IFA.
The lamin is localized at the nuclear periphery and the lamina by indirect immunofluorescence. Immunogold labelling of nuclear matrix sections reveals that the protein is not only associated with the lamina, but also with the internal matrix. Taken together these results reveal that higher plants, which do not possess an organized network of cytoplasmic intermediate filaments, nevertheless present a well-organized lamina containing lamins in which at least one of them is immunologically related to vertebrate lamin B. Our data confirm that lamins are very old members of the intermediate filament proteins that have been better conserved in plants during evolution than their cytoplasmic counterparts.
The 270 kda splice variant of erythrocyte β-spectrin (βi∑2) segregates in vivo and in vitro to specific domains of cerebellar neurons
Authors: Malchiodi-Albedi Fiorella , Ceccarini Marina , Winkelmann John C. , Morrow Jon S. , Petrucci Tamara C. 
Published: April 2021

ABSTRACT
Spectrin isoforms arise from four distinct genes, three of which generate multiple alternative transcripts. With no βIochemical restrictions on the assembly of αβ het-erodimers, more than 25 distinct heterodimeric spectrin species may exist. Whether (and why) this subtle but substantial diversity is realized in any single cell is unknown. To address this question, sequence-specific antibodies to alternatively spliced regions of α- and β-spectrin have been prepared. Reported here is the localization in rat cerebellar neurons at light and electron microscopic levels of an antibody against a unique sequence (βI∑2-A = PGQHKDGQKSTGDERPT) from the 270 kDa transcript of the red cell β-spectrin gene (spectrin I 2). In this version, the 3 sequence of ery-throid β-spectrin (βI∑1) is replaced with an alternative sequence that shares substantial homology with the 3’ sequence of non-erythroid -β-spectrin (βI∑1). The antibody to βI∑2-A stains a single protein band at 270 kDa, determined by western blotting, in both rat cerebellum and in cultured cerebellar granule cells, and does not react with βII∑1 spectrin (β-fodrin). This antibody stains the dendritic spines of Purkinje cells in the molecular layer, and is concentrated at postsynaptic densities (PSDs) adjacent to synapsin I (which is confined to the presynaptic membrane). The soma of Purkinje cells do not stain. In the granular layer, cytoplasmic organelles and the postsynaptic densities of granular cells stain strongly. Astrocytes are also stained. In all cells, plasma membrane staining is confined to postsynaptic densities (PSD). The βI∑2 isoform co-immunoprecipitates with non-erythroid -spectrin (αII∑2*), even though the distribution of II within neurons only partially overlaps that of βI∑2 No hybrid βI∑2 and βII∑1 (β-fodrin) spectrin complexes appear to exist. Spectrin βI∑2 is also polarized in cultured rat cerebellar granule cells, where it is abundant in cell bodies but not neurites. The overall distribution of βI∑2 is as a subset of the distribution of spectrins 240/235E previously detected with a generally reactive erythrocyte αβspectrin antibody. These findings establish the highly precise segregation of a β-spectrin isoform to distinct cytoplasmic and membrane surface domains, indicate that it is complexed (partially) with non-erythroid - spectrin, and demonstrate that cytoskeletal targeting mechanisms are preserved in cultured granular cells. The extreme concentration of βI∑2 spectrin at the PSD and in selected cytoplasmic compartments suggests that unique isoforms of spectrin may play a pivotal role in organizing topographically defined clusters of receptors or cytoplasmic protein complexes.
Interactions between neutrophils and endothelial cells
Authors: Godin Christine , Caprani Adrien , Dufaux Jacques , Flaud Patrice 
Published: April 2021

ABSTRACT
Independently of their protective influence, leukocytes can have harmful effects in pathological situations such as diabetes (Benhamou et al., 1990), ischemia-reperfusion injury (Hernandez et al., 1987; Schmid-Schönbein and Engler, 1990; Thornton et al., 1989), inflammatory disorders (Fiebig et al., 1991; Grant, 1973; Jutila, 1992) and vascular permeability disorders (Kubes et al., 1991a; Schoenberg et al., 1991). Their action is mainly due to a rolling phenomenon along venular walls, adhesion to vascular endothelial cells and migration across the endothelium. A number of factors govern the interactions between leukocytes and endothelial cells, including the expression of molecules on the surface of activated leukocytes and/or endothelial cells, certain products of neutrophil activation, and shear forces within blood vessels that tend to sweep leukocytes along the endothelial cell surface (Harlan, 1985; Jutila, 1992; Ley, 1989; Tonnesen, 1989; Zimmerman et al., 1992).
Is the gap junction channel - The Connexon - made of connexin or ductin?
Authors: Finbow Malcolm E. , Pitts John D. 
Published: April 2021

ABSTRACT
“For a‘ that, an a’ that
Our toils obscure an a’ that”
Robert Burns
The liver gap junction and Halobacterium purple membrane were among the first organised membrane complexes to be isolated. The retention of their paracrystalline form during isolation provided opportunities for physical analysis and allowed early glimpses of the structural organisation of integral membrane proteins. The seminal study of Henderson and Unwin (1975) on the purple membrane, using electron diffraction analysis and image reconstruction, revealed a detailed topology of the subunits that could be related to the structure of the component protein, bacteriorhodopsin. The work provided a framework for understanding the helical nature of the protein within the membrane bilayer and this model now serves as a structural archetype for a super-family of proteins with seven transmembrane α-helices. Other integral membrane proteins have now been crystallised that confirm the suggestion from Henderson and Unwin’s study that the secondary structure and packing fea-tures of such proteins are very similar to those of their water-soluble counterparts.
The differentiation and lineage development of goblet cells in the murine small intestinal crypt: experimental and modelling studies
Authors: Paulus Ursula , Loeffler Markus , Zeidler Joachim , Owen Gary , Potten Christopher S. 
Published: April 2021

ABSTRACT
The objective of this study was to provide a new insight into the origin and lineage development of mucus-producing cells in the small intestinal crypt. For this, new experimental data were obtained from both crypt sections and whole mounts. Model simulation studies were undertaken to investigate which rules are most likely to govern the dynamic cellular development and goblet cell pedigree.
We have measured the frequency of mucus-secreting goblet cells (using alcian blue and periodic acid Schiff’s stains) at each cell position in the ileal murine crypt. These measurements, made on sections, overestimate the number of goblet cells because of the size and centripetal position of the stained cytoplasm. The correction factor for this overscoring has been measured to be 0.25 by two independent methods. The data suggest that there are about 12 functional goblet cells per crypt many of which retain an ability to divide. We have also determined the labelling index of the crypt goblet cells at each cell position. Spatially, goblet cells exhibit a small degree of clustering in the crypt and show a good mixture with columnar cells.
We have adapted our earlier dynamic matrix-based computer simulation model to take into account goblet cell differentiation. The modelling suggested the following conclusions: firstly, goblet cells do not have their own stem cells but share a common stem cell with the columnar cells; secondly, the goblet lineage differentiates from the transit population two to three generations before the end of the lineage; and thirdly, the decision to switch on goblet properties is stochastic at a specific step in the development of columnar cells.
Lipid and membrane protein transfer from human neutrophils to schistosomes is mediated by ligand binding
Authors: Rogers Rick A. , Jack Richard M. , Furlong Stephen T. 
Published: April 2021

ABSTRACT
Attachment of human neutrophils to schistosomula of Schistosoma mansoni involves leukocyte receptors recognizing carbohydrate, complement and/or IgG ligands on the parasite surface. Here, we examined the transfer of a fluorescent fatty acid analog (BOFA) from human neutrophils to schistosomula coated with concanavalin A (Con A), immune serum or nonimmune serum under co-culture conditions by fluorescence confocal laser scanning microscopy (CLSM). Coating schistosomes with Con A or immune serum and co-culturing them for 24 hours with BOFA-labeled neutrophils resulted in a specific lipid transfer to the surface tegument of the schistosomes. Tegumental labeling was absent when nonimmune serum was used. No significant difference (P&lt;0.001) was found in the number of neutrophils bound to the worm surface between Con A-coated schis-tosomes (4.1 ± 0.345 cells/worm) and worms incubated in immune serum (4.261 ± 0.362). The number of neutrophils bound to the schistosomula (2.7 ± 0.223) was significantly reduced in the presence of nonimmune serum (P&lt;0.0001). The viability of the schistosomula was 98% in nonimmune treated co-cultures, and 91% in co-cultures treated with immune serum. HPLC analysis of labeled neutrophils demonstrated that BOFA was incorporated into both phospholipids and neutral lipids, which were almost exclusively triglycerides and, after 18 hours of culture, all of the fatty acid analog was incorporated into complex lipids. Double-label experiments in which schistosomula bearing Con A were first incubated with BOFA-labeled neutrophils and subsequently immunolabeled revealed that the neutrophil membrane proteins, MHC class I, CR1 and CR3 were co-transferred with neutrophil lipids to the parasite tegument. Thus, the parasite acquires necessary lipids as well as host membrane proteins in a native conformation that may play a role in parasite protection via mimicry (class I), regulation of complement activation (CR1) and binding of host integrin ligands (CR3).
Regulation of cd18 expression in human neutrophils as related to shape changes
Authors: Volz Alexandra 
Published: April 2021

ABSTRACT
The study analyses the distribution and quantitative expression of surface CD18 of neutrophils exposed to distinct stimuli that produce different types of continuous shape changes, including types that are associated with locomotion and others that are not. The chemotactic peptide N-formyl-L-norleucyl-L-leucyl-L-phenylalanine, colchicine and nocodazole were used to induce a polarized locomotor morphology, phorbol myristate acetate, 1,2-dioctanoylglycerol and 1-oleoyl-2-acetylglycerol to induce non-polar motile cells ruffling all over the surface and 2H2O to induce non-polar cells performing circus movements as have been previously described. Except for colchicine and nocodazole, these stimuli increased surface expression of CD18. Thus, stimulated shape changes are frequently, though not always, associated with increased surface expression of CD18. High concentrations (10 7 to 10 5 M) of phorbol myristate acetate but not of chemotactic peptide induced down-regulation of surface CD18. Cytochalasin D (10 4 M) stimulated CD18 expression even though it inhibited shape changes. The surface distribution of CD18 determined by light microscopy was uniform in unstimulated cells or in various forms of stimulation except for cells treated with 10 5 M cytochalasin D. Cytochalasin D (10 5 M) produced CD18 accumulation at the pole opposite the F-actin cap. Experiments with colchicine, nocodazole, 2H2O and cytochalasin D suggest that microtubules as well as microfilaments modulate surface expression of CD18.
The results suggest that protein kinase C and phosphatases play a role in regulating surface expression of CD18 in neutrophils. Increased CD18 expression in response to phorbol myristate acetate, but not in response to chemotactic peptide was inhibited by the protein kinase C inhibitor Ro 31-8220 (10 6 to 10 5 M). Ro 31-8220 (10 5 M) alone increased CD18 expression of initially resting cells. Furthermore, the phosphatase inhibitor okadaic acid (5 M) and calyculin A (0.04 μM) suppressed CD18 expression in chemotactic peptide-treated cells, but there was no detectable effect in phorbol myristate acetate-stimulated cells. This indicates that at least two different pathways mediating CD18 expression are operative in human neutrophils, one being protein kinase C-dependent, the other(s) probably being protein kinase C-independent.
Calcium buffer injections inhibit cytokinesis in Xenopus eggs
Authors: Miller Andrew L. , Fluck Richard A. , McLaughlin Jane A. , Jaffe Lionel F. 
Published: April 2021

ABSTRACT
A slow cortical wave of high calcium accompanies the elongation of cleavage furrows in medaka fish eggs as well as in Xenopus eggs. We explored the role of such waves by injecting calcium buffers into Xenopus eggs at various times before and during first and second cleavage. Injection earlier than about 15 minutes before first cleavage normally starts delays it for hours. Injection between about 15 minutes and a few minutes before cleavage normally starts allows a (short) furrow to form on time but usually yields an eccentric one. This forms away from the injection side, often as far off-center as the egg’s equator, and then regresses. Injection soon after it starts quickly arrests elongation of the furrow and eventually induces its regression; while injection a bit later likewise soon arrests elongation but allows delocalized furrow deepening to continue. The dependence of these inhibitory actions upon the dissociation constants and final cytosolic concentrations of the injected buffers indicates that they act as shuttle buffers to suppress needed zones of high calcium in the micromolar range. We conclude that the high calcium that is found within these furrows is needed to induce them, to extend them and even to maintain them.
Moreover, while short, eccentric furrows often form as far off center as the equator, they somehow always form along a meridian through the animal pole. This seems difficult to explain by the orthodox, diastral model. Rather, it suggests that the cleavage furrows in Xenopus- and perhaps in animal cells quite generally- are directly induced by a diastema or telophase disc rather than by the asters.
Nuclear localization of vertebrate cyclin a correlates with its ability to form complexes with cdk catalytic subunits
Authors: Maridor G. , Gallant P. , Golsteyn R. , Nigg E. A. 
Published: April 2021

ABSTRACT
Cyclins control the activities of cyclin-dependent protein kinases (cdks) and hence play a key role in cell cycle regulation. While B-type cyclins associate with p34cdc2 to trigger entry into mitosis, progression through S phase requires cyclin A, presumably in association with p33cdk2. Vertebrate A- and B-type cyclins display strikingly distinct subcellular localizations, but the mechanisms underlying these differential distributions are unknown. Here, we have begun to study the requirements for nuclear localization of cyclin A. We have isolated a cDNA coding for chicken cyclin A and constructed a series of deletion mutants. These were then transfected into HeLa cells, and the subcellular distribution of the mutant cyclin A proteins was determined by indirect immunofluorescence microscopy. In parallel, the cyclin A mutants were assayed for their ability to form complexes with cdk subunits. We found that deletion of more than 100 residues from the N terminus of cyclin A did not impair nuclear localization or cdk subunit binding and kinase activation. In contrast, removal of as few as 15 residues from the C terminus, or deletion of part of the internal cyclin box domain, abolished nuclear localization of cyclin A as well as its ability to bind to and activate cdk subunits. These results suggest that nuclear transport of cyclin A may depend on the formation of multiprotein complexes comprising cdk catalytic subunits.
Cloning of the polyubiquitin cDNA from the marine sponge Geodia Cydonium and its preferential expression during reaggregation of cells
Authors: Pfeifer Karin , Frank Wolfgang , Schröder Heinz C. , Gamulin Vera , Rinkevich Baruch , Batel Renato , Müller Isabel M. , Müller Werner E. G. 
Published: April 2021

ABSTRACT
Ubiquitination of proteins is a critical step in the controlled degradation process of many polypeptides. Here we show that sponges, the simplest multicellular group of eukaryotic organisms, are also equipped with the ubiquitin pathway. The polyubiquitin cDNA was isolated and characterized from the marine sponge Geodia cydonium. The open reading frame contains six ubiquitin moieties, which are lined up head to tail without spacers. A comparison of the predicted amino acid sequence of the six sponge ubiquitin-coding units with those from other organisms revealed a high degree of homology (&gt; 93%). The ubiquitin gene is expressed to almost the same extent in the two main compartments of the sponge, the cortex and the medulla. However, only in the cortex are detectable amounts of the ubiquitin protein synthesized. The ubiquitin protein isolated from the sponge organism was found to initiate protein degradation in the heterologous reticulocyte system in the same manner as bovine ubiquitin. In vitro studies with dissociated sponge cells revealed that the homologous aggregation factor causes (i) a strong increase in the steady-state level of mRNA coding for ubiquitin and (ii) a drastic increase in ubiquitin protein synthesis, while the homologous lectin failed to display that effect in isolated cells. These data suggest that ubiquitin may play a role in sponge morphogenesis.
An isoform of microtubule-associated protein 2 (MAP2) containing four repeats of the tubulin-binding motif
Authors: Doll T. , Meichsner M. , Riederer B. M. , Honegger P. , Matus A. 
Published: April 2021

ABSTRACT
Microtubule-associated protein 2 (MAP2) exists in both high- and low-molecular mass isoforms, each of which has a tubulin-binding domain consisting of 3 imperfect tandem repeats of 31 amino acids containing a more highly conserved 18 amino acid ‘core’ sequence. We describe here a novel form of low molecular mass MAP2 (MAP2c) that contains an additional 4th repeat of this tubulin-binding motif. Like the 3 previously known repeat sequences, this 4th copy is highly conserved between MAP2 and the two other known members of the same gene family, tau and MAP4. In each of these three genes the additional 4th repeat is inserted between the 1st and 2nd repeats of the 3-repeat form of the molecule. Experiments with brain cell cultures, in which the relative proportions of neurons and glia had been manipulated by drug treatment, showed that 4-repeat MAP2c is associated with glial cells whereas 3-repeat MAP2c is expressed in neurons. Whereas 3-repeat MAP2c is expressed early in development and then declines, the level of 4-repeat MAP2c increases later in development, corresponding to the relatively late differentiation of glial cells compared to neurons. When transfected into non-neuronal cells, the 4-repeat version of MAP2c behaved indistinguishably from the 3-repeat form in stabilising and rearranging cellular microtubules. The presence of an additional 4th repeat of the tubulin-binding motif in all three members of the MAP2 gene family suggests that this variant arose prior to their differentiation from an ancestral gene.