bluuebunny/crossref_metadata_2025_split huggingface dataset:
The macrophage capacity for phagocytosis
Authors: Cannon Gregory J. , Swanson Joel A. 
Published: April 2021

ABSTRACT
Murine bone marrow-derived macrophages, which measure 13.8 ± 2.3 tun diameter in suspension, can ingest IgG-opsonized latex beads greater than 20 μm diameter. A precise assay has allowed the determination of the phagocytic capacity, and of physiological parameters that limit that capacity. Ingestion of beads larger than 15 μn diameter required IgG-opsonization, and took 30 minutes to reach completion. Despite the dependence on Fc-receptors for phagocytosis of larger beads, cells reached their limit before all cell surface Fc-receptors were occupied. The maximal membrane surface area after frustrated phagocytosis of opsonized coverslips was similar to the membrane surface area required to engulf particles at the limiting diameter, indicating that the capacity was independent of particle shape. Vacuolation of the lysosomal compartment with sucrose, which expanded endocytic compartments, lowered the phagocytic capacity. This decrease was reversed when sucrose vacuoles were collapsed by incubation of cells with invertase. These experiments indicate that the phagocytic capacity is limited by the amount of available membrane, rather than by the availability of Fc-receptors. The capacity was also reduced by depolymerization of cytoplasmic microtubules with nocodazole. Nocodazole did not affect the area of maximal cell spreading during frustrated phagocytosis, but did alter the shape of the spread cells. Thus, microtubules may coordinate cytoplasm for engulfment of the largest particles.
Behaviour of ejaculated spermatozoa from bull, boar and ram during thin-layer countercurrent partition in aqueous two-phase systems
Authors: Harrison R. A. P. , Jacques M. L. , Minguez M. L. Pascual , Miller N. G. A. 
Published: April 2021

ABSTRACTEjaculated spermatozoa from bulls, boars and rams were subjected to thin-layer countercurrent partition in aqueous two-phase systems composed of dextran T500 and polyethylene glycol 6000 (PEG) in sucrose-based Hepes-buffered media. In the basal system, the great majority of spermatozoa tended to partition with the dextran-rich bottom phase; however, by including very low levels of phosphate, they could be made to partition increasingly with the PEG-rich top phase (complete at 10 mM phosphate). A procedure was developed for carrying out four separations simultaneously under identical conditions, whereby it could be shown that distribution varied with the number of spermatozoa in the sample. In the case of bull, the effect of cell number could be reduced considerably by inclusion of small quantities of seminal plasma in the phase system, but no such effect was found for ram or boar. Considerable variation in distribution pattern was seen between samples, which did not appear to be due to technical inconsistency. Livability in the phase systems was also variable, and we believe that PEG may exert a detergent-like effect on the sperm surface that is exacerbated in highly defined media free of protective proteins.
Identification of the intermediate filament-associated protein gyronemin as filamin: Implications for a novel mechanism of cytoskeletal interaction
Authors: Brown Kevin D. , Binder Lester I. 
Published: April 2021

ABSTRACT
In a previous paper, a monoclonal antibody (designated Ml.4) that recognized a 240 kDa polypeptide was characterized. This antibody stained the intermediate filaments in several cell lines, and biochemical characteristics of the 240 kDa polypeptide led us to conclude that it was a novel intermediate filament-associated protein, which we termed gyronemin. Here we report that gyronemin is expressed in adult rat organs that contain a substantial smooth muscle component. Taking advantage of this observation, this protein was purified from bovine uterine tissue and, by biochemical, immunological and amino acid sequence analysis, found to be homologous to the actin-associated protein filamin. Three novel monoclonal antibodies raised using purified bovine gyronemin as the immunogen show this protein to be associated with actin-containing stress fibers, although our original Ml.4 antibody continued to be localized along vimentin filaments. Since two-dimensional electrophoretic analysis did not demonstrate a difference in either relative molecular mass or isoelectric point of this polypeptide when associated with either filamentous system, we conclude that filamin is a bifunctional protein capable of associating with both the intermediate filament and actin cytoskeletal systems.
Involvement of the consensus sequence motif at coil 2b in the assembly and stability of vimentin filaments
Authors: Kouklis Panos D. , Traub Peter , Georgatos Spyros D. 
Published: April 2021

ABSTRACT
Nearly all intermediate filament (IF) proteins share two sequence motifs located at the N- and the C-terminal ends of their helical rod domain (‘coil la’ and ‘coil 2b’, respectively). To examine the structural role of the coil 2b motif, we have performed in vitro assembly studies and in vivo microinjection experiments employing two site-specific reagents: (a) a 20-residue synthetic peptide (C-2) representing the conserved motif itself and (b) a monoclonal antibody (anti-IFA) that recognises an epitope within the conserved coil 2b sequence. We demonstrate here that vimentin protofilaments, when induced to assemble in the presence of C-2 or anti-IFA, show a lower propensity to polymerise and yield various abberant structures. The few filaments that are formed under these conditions appear much shorter than normal IFs and are unravelled or aggregated. Furthermore, when preformed vimentin filaments are exposed to C-2 or anti-IFA, most of the normal IFs are converted into shorter filamentous forms that possess an abberant morphology. None of these effects is seen when vimentin subunits are coincubated with control peptides. Microinjection of anti-IFA into the cytoplasm of interphasic 3T3 cells provokes collapse of vimentin IFs into a juxtanuclear mass and formation of numerous amorphous aggregates distributed throughout the cytoplasm. These two effects are not seen when the anti-IFA is microinjected into the cell nucleus. Our results provide experimental evidence supporting previous suggestions for a role for the conserved coil 2b sequence in filament assembly. We propose that this region is interacting with other sites along the vimentin molecule and that these interactions are essential for proper protofilament-protofilament alignment and filament stability.
Association of cyclin-bound p34cdc2 with subcellular structures in Xenopus eggs
Authors: Leiss D. , Félix M.-A. , Karsenti E. 
Published: April 2021

ABSTRACT
Cell cycle progression is controlled by changes in kinase activity of homologs of the fission yeast protein p34cdc2. The p34cdc2 kinase is activated by its association with a cyclin subunit, followed by post-translational modifications. Here, we show that in Xenopus eggs stimulated to enter the early embryonic cell cycle by an electric shock, part of the p34cdc2 becomes associated with subcellular fractions as the eggs progress towards mitosis. This occurs as a result of cyclin accumulation because most of the B-type cyclins and some of the A-type cyclins are found in the particulate fraction. Moreover, as soon as cyclins are degraded, p34cdc2 is released in the soluble fraction. The p34cdc2-cyclin complex can be solubilised by 80 mM β-glycerophos-phate (in the standard MPF extraction buffer) or by high salt concentrations. The post-translational modifications leading to cdc2 kinase activation by cyclin occur in the insoluble form. Following fractionation of egg extracts by sucrose gradient centrifugation, the p34cdc2 -cyclin B complex is found in several fractions, but especially in two discrete peaks. We present evidence that in the slow-sedimenting peak the p34cdt2-cyclin B complex is associated with the 60 S subunit of monoribosomes. It could be targeted in this fashion to substrates such as ribosomal proteins and maybe to cytoskeletal proteins, since ribosomes bind to microtubules and are present in the spindle. The p34cdc2-cyclin B complex is also found in a faster-migrating fraction containing various membranous structures, including Golgi stacks. Therefore, as observed by immunofluorescence in other systems, it seems that cyclin subunits target p34cdc2 to specific cellular sites and this is certainly important for its function. In addition, we present preliminary evidence suggesting that some component present in the ribosome-containing fraction is required for activation of the p34cdc2-cyclin B complex.
Adhesion of 8701-bc breast cancer cells to type v collagen and 67 kda receptor
Authors: Minafra Salvatore , Luparello Laudio , Pucci-Minafra Ida , Sobel Mark E. , Garbisa Spiridione 
Published: April 2021

ABSTRACT
Ductal infiltration carcinomas (d.i.c.) of the breast are potentially highly metastatic tumours, associated with drastic alterations of the architecture and molecular composition of the extracellular matrix at the tumourhost interface. 8701-BC, a recently characterized cell line, isolated from primary d.i.c., was used to study different aspects of tumor cell-substratum interactions. Since type V collagen deposition is augmented in d.i.c. we have examined the ability of 8701-BC cells to interact with this collagen species. We have found that cell binding to type V collagen was mediated by protein homologous to the 67 kDa laminin receptor (67-R). This conclusion is substantied by the following observations: (a) a major band having an apparent molecular mass of 67 kDa and immunoreactive to the anti-67 R antibody was detectable by SDS-PAGE of the membrane proteins; (b) the antibody inhibited cellular adhesion to type V collagen in a dose-dependent way; (c) membrane proteins purified by affinity chromatography on type V collagen were immunoreactive to anti-67 R antibody, but not to anti-VL Al, VLA2 and VLA3 integrin antibodies. This receptor appears to have prominent carbohydrate-binding properties, since lactose competes with cell adhesion to type V collagen.
Differential lectin binding to presumptive cortical cells of the wool follicle bulb
Authors: Campbell Mary T. , Dollin Anne E. , Adelson David L. 
Published: April 2021

ABSTRACT
An a-D-galactoside-specific lectin from Bandeiraea sim-plicifolia (BSLI) showed differential binding to cortical cells of the wool follicle bulb. The lectin bound to cells on one side only of the bulb and was completely blocked by a-D-galactose. The region of lectin binding extended from presumptive cortical cells at the base of the bulb to cortical cells at the top of the bulb, disappearing as cortical cells entered the fibre cortex. An orthocortexspecific monoclonal antibody was used to show that cortical cells recognised by the lectin lie directly below the fibre orthocortex and presumably give rise to the orthocortex. The results suggest that two distinct populations of presumptive cortical cells are present only two to three cell layers from the base of the bulb in a region where no morphological differences are detectable. The lectin-bound pre-cortical cells appear to give rise to orthocortical cells while cells not bound by lectin presumably give rise to paracortical cells.
Electron microscopy showed that the lectin bound to sites on the plasma membrane, probably on the extracellular surface. This suggests that the lectin target may be a membrane glycoprotein or glycolipid. Two polypeptides recognised by BSLI were separated from wool follicle extracts by SDS-gel electrophoresis. These polypeptides migrated at approximately 69 kDa and 17 kDa. However, only the 69 kDa molecule showed the expected loss of binding by BSLI in the presence of a-D-galactose. Further work will be required to determine if this glycoprotein is the bulb cell molecule recognised by BSLI. Dermal papilla cells were also bound by BSLI and may carry the same, or a similar, cell-surface molecule to presumptive orthocortical cells.
BSLI appears to be a useful marker for detecting patterns of cell differentiation in the bulb that correlate with patterns of cortical cell type in the wool fibre. The lectin may also be a useful tool for distinguishing cortical cells of different lineage in cell culture. Furthermore, the pattern of BSLI binding seen in small follicle bulbs appears to provide some support for a reaction-diffusion model of follicle differentiation.
Phosphorylated baculovirus p10 is a heat-stable microtubule-associated protein associated with process formation in Sf9 cells
Authors: Cheley Stephen , Kosik Kenneth S. , Paskevich Peter , Bakalis Shelley , Bayley Hagan 
Published: April 2021

ABSTRACT
Insect ovarian Sf? cells extend processes with complex morphologies when infected with a recombinant baculovirus encoding the catalytic subunit of protein kinase A. Within the shafts of the processes are abundant microtubules, which, in contrast to those in Sf9 cells expressing the microtubule-associated protein tau, are generally not organized into parallel bundles. During infection the late viral polypeptide p10 becomes phosphorylated by the protein kinase A catalytic subunit at its penultimate residue, Ser92. The expression or phosphorylation of other major host cell or viral polypeptides does not change, compared with polypeptides from a wild-type viral infection. Once phosphorylated, p10 associates with microtubules in the infected cells and may thereby play a role in process formation.
A gene family consisting of ezrin, radixin and moesin Its specific localization at actin filament/plasma membrane association sites
Authors: Sato Naruki , Funayama Noriko , Nagafuchi Akira , Yonemura Sfugenobu , Tsukita Sachiko , Tsukita Shoichiro 
Published: April 2021

ABSTRACT
Radixin is a barbed end-capping actin-modulating protein which was previously reported to be concentrated at cell-to-cell adherens junctions (AJ) and cleavage furrows. Recently, cDNA encoding mouse radixin was isolated, showing that radixin is highly homologous to but distinct from ezrin. From mouse teratocarcinoma cells we isolated and analyzed cDNA encoding another radixin-related protein. Sequence analysis has demonstrated that this protein is a mouse homologue of human moesin (98.3% identity) and that it shares 71.7% and 80.1% identity with ezrin and radixin, respectively. Translation experiments in vitro combined with immunoblot analyses led us to conclude that there is a gene family consisting of ezrin, radixin and moesin. These members are coexpressed in various types of cells. Then, by immunofluorescence microscopy, we closely analyzed their distribution using polyclonal and monoclonal antibodies, which could recognize all three members. In addition to cell-to-cell AJ and cleavage furrows, it was shown that they were concentrated at microvilli and ruffling membranes in various types of cells. Furthermore, the cell-to-substrate AJ (focal contacts) were clearly stained by anti-radixin pAb only after the apical/lateral membranes and cytoplasm were removed by the zinc method. We conclude that at least one of the members of the ezrinradixin-moesin family is concentrated at specific regions where actin filaments are densely associated with plasma membranes.
BN46/51, a new nucleolar protein, binds to the basal body region inNaegleria gruberiflagellates
Authors: Trimbur Gina M. , Walsh Charles J. 
Published: April 2021

ABSTRACTIndirect immunofluorescence with the monoclonal antibody, BN5.1, labels the nucleolus of amebae of the amebo-flagellate Naegleria gruberi. When amebae differentiate into flagellates, BN5.1 binds to nucleoli and to the basal body region. The BN5.1 antigen is not present in basal bodies when basal bodies form at about 60 min after initiation of the differentiation or when flagella form at about 70 min. The BN5.1 antigen is first detectable in the basal body region at 85 min after initiation, a time when the basal body region acts as a microtubule organizing center for the formation of the microtubule cytoskeleton (MTCS) of flagellates. When flagellates revert spontaneously to amebae, the BN5.1 antigen is lost from the basal body region coincident with the loss of the MTCS. The BN5.1 antigen, composed of approximately equal amounts of two subunits of 46 kDa and 51 kDa, both of which carry the BN5.1 epitope, has been named BN46/51. BN46/51 in the basal body region comigrates with the nucleolar antigen by two-dimensional gel electrophoresis. Approximately 75% of the nucleolar BN46/51 is solubilized by extracton with 0.4 M NaCl. However, the antigen associated with the basal body region is resistant to extraction, even with 2 M NaCl. Solubilized BN46/51 exists as a heterogeneous multimer that elutes on gel filtration with a peak at 400 to 500 kDa and sediments on sucrose gradients at 5.5 S. The multimers consist of only the 46 kDa and the 51 kDa subunits in equal amounts as judged by glutaraldehyde cross-linking and by chromatography on BN5.1 affinity columns. Nucleolar BN46/51 is associated with the dense fibrillar and granular components of the nucleolus. However, it does not resemble any previously described nucleolar protein. Neither BN5.1, nor three other mAbs that recognize additional epitopes on both the 46 kDa and 51 kDa subunits of BN46/51, binds to nucleoli from Saccharomyces cerevisiae or mammalian cells. BN5.1 does not bind to the nucleoli of Dictyostelium discoideum or Euglena gracilis. Thus BN46/51 is an unusual and perhaps unique nucleolar component whose presence in the basal body region presents a challenge to our understanding of the cytoskeleton.
Association of myosin with the connecting cilium of rod photoreceptors
Authors: Williams David S. , Hallett Mark A. , Arikawa Kentaro 
Published: April 2021

ABSTRACT
The cilium of a vertebrate photoreceptor cell connects the phototransductive outer segment of the cell to the inner segment. Previous studies have shown that, within the connecting cilium, there is a small cluster of actin filaments, which play a critical role in the formation of new disk membranes. Here, we have detected a polypeptide in rat rod outer segments that is recognized by myosin heavy chain antibodies and was found to possess other characteristics of conventional non-muscle myosin heavy chain: it comigrates in SDS-PAGE with non-muscle myosin heavy chain; it associates with the cytoskeleton of rod outer segments in an ATP-sensitive manner; and it binds to purified actin filaments in the absence of ATP. Myosin ATPase activity was also detected in isolated rod outer segments. Electron immunomicroscopy revealed that myosin is present in the small actin-containing domain within the connecting cilium at the site of disk membrane morphogenesis. These results pose the possibility that an actin-myosin contractile mechanism functions in the formation of new photoreceptor disk membranes.
Endocytosis: what goes in and how?
Authors: Watts Colin , Marsh Mark 
Published: April 2021

ABSTRACT
During the last decade the term ‘endocytosis’ has become virtually synonymous with the activity of clathrin-coated vesicles. These vesicles, which are derived from cell surface clathrin-coated pits, are transport vehicles responsible for the transfer of plasma membrane receptors and their ligands, between the first two stations of the endocytic pathway: namely, the plasma membrane and early endosomes (Goldstein et al., 1985; van Deurs et al., 1989; Griffiths and Gruenberg, 1991). Despite the irrefutable evidence that clathrin-coated vesicles mediate endocytosis, their contribution to the total endocytic activity of the cell and the composition of the membrane they internalise remains controversial. Here we discuss: (1) the evidence that non-clathrin-mediated endocytic mechanisms operate alongside the clathrin-mediated pathway; (2) the evidence that endocytosis occurs for surface molecules that are not enriched in clathrin-coated pits and; (3) the sorting activities of cell surface clathrincoated pits and the notion that plasma membrane proteins that show particularly slow rates of uptake are actively excluded from the endocytic pathway.
Characterization of early and late endocytic compartments of the transferrin cycle Transferrin receptor antibody blocks erythroid differentiation by trapping the receptor in the early endosome
Authors: Killisch Iris , Steinlein Peter , Römisch Karin , Hollinshead Ruth , Beug Hartmut , Griffiths Gareth 
Published: April 2021

ABSTRACT
We describe a detailed morphological characterization of the endocytic pathway in differentiating chicken erythroblasts transformed by a temperature-sensitive mutant of avian erythroblastosis virus (AEV). These cells express high levels of transferrin receptors (TfR) when induced to differentiate at 42°C. Biochemical analysis showed that most (≈90%) of the internalized 125I-Tf recycled within ≈30 min while a smaller fraction of l25I-Tf required up to 2 h for recycling. By immunocytochemistry, the bulk of Tf and TfR was localized at the plasma membrane and in tubuloreticular early endosomes. This structure contained coated buds that labelled with an antibody specific for the clathrin light chain. Decreasing amounts of both Tf and TfR were detected in two distal compartments, spherical endosome vesicles resembling multivesicular bodies and the prelysosomal compartment (PLC) enriched in cation-independent mannose 6-phosphate receptor. As shown by fluorescent (F1TC-Tf) labelling of living cells, the movement of Tf/TfR complex into these late structures was accompanied by a significant drop in pH from about 6, the value displayed by early endosomes, to values below pH 5.0. Since no detectable 125I-Tf degradation was observed during a 4 h period we believe that the Tf/TfR detected in these late endocytic structures avoids degradation and recycles back to the cell surface.
The addition of an anti-TfR monoclonal antibody to the culture medium of these cells blocks their differentiation. Under this condition the antibody-TfR complex was trapped in an early endosome compartment that enlarged to more than twice its normal size. However, this condition did not affect the transport kinetics of horseradish peroxidase from the medium to the PLC.
Dimeric transcription factor families: it takes two to tango but who decides on partners and the venue?
Authors: Lee Kevin A. W. 
Published: April 2021

ABSTRACT
Dimeric transcription factors that bind to DNA are often grouped into families on the basis of dimerization and DNA-binding specificities. cDNA cloning studies have established that members of the same family have structurally related dimerisation and DNA-binding domains but diverge in other regions that are important for transcriptional activation. These features lead to the straightforward suggestion that although all members of a family bind to similar DNA elements, individual members exhibit distinct transcriptional effector functions. This simple view is now supported by experimental evidence from those systems that have proved amenable to study. There are however some largely unaddressed questions that concern the mechanisms that allow family members to go about their business without interference from their highly related siblings. Here I will discuss some insights from studies of the bZIP class of transcription factors.