bluuebunny/crossref_metadata_2025_split huggingface dataset:
Basolateral secretion of kappa light chain in the polarised epithelial cell line, Caco-2
Authors: Hughson Elaine J. , Cutler Daniel F. , Hopkins Colin R. 
Published: April 2021

ABSTRACT
The immunoglobulin kappa light chain is constitutively secreted in non-polarised cells. It is therefore unlikely to display any of the signals thought to be required for the selective delivery of proteins to the apical or basolateral borders of polarised epithelial cells. We have transfected the gene for the kappa light chain into a polarised epithelial cell line (Caco-2) and shown that it is secreted predominantly from the basolateral surface. Metabolically labelled endogenous secretory products show the same polarity and we conclude, therefore, that in Caco-2 cells there is a major intracellular trafficking route to the basolateral border that requires no sorting signal.
An ultrastructural study of crossbridge arrangement in the fish skeletal muscle thick filament
Authors: Kensler Robert W. , Stewart Murray 
Published: April 2021

ABSTRACT
A procedure has been developed for isolating goldfish skeletal muscle thick filaments that preserves the near-helical arrangement of the myosin crossbridges under relaxing conditions. These filaments have been examined by electron microscopy and computer image analysis. Electron micrographs of the negatively stained filaments showed a clear periodicity associated with the crossbridges, with an axial repeat every 42.9 nm. Computed Fourier transforms of the negatively stained filaments showed a series of layer lines confirming this periodicity, and were similar to the X-ray diffraction patterns of fish muscle obtained by J. Harford and J. Squire. Analysis of the computed transform data and filtered images of the isolated fish filaments demonstrated that the myosin crossbridges lie along three strands. Platinum shadowing demonstrated that the strands have a right-handed orientation, and computed transforms and filtered images of the shadowed filaments suggest that the crossbridges are perturbed both axially and azimuthally from an ideal helical arrangement.
The behaviour of microtubules in chromosomal spindle fibres irradiated singly or doubly with ultraviolet light
Authors: Wilson Paula , Forer Arthur 
Published: April 2021

ABSTRACT
Areas of reduced birefringence (ARBs) produced by ultraviolet microbeam irradiation are areas of depolymerized microtubules. ARBs probably move poleward either by microtubule subunit addition at the kinetochore and loss at the pole, or by microtubule subunit addition at one edge of the ARB and loss from the other edge. In this paper we have used two approaches to try to distinguish between these two models. First, we determined whether the edges of the ARB move at the same rate; if ARB motion is due solely to addition at the kinetochore and loss at the pole, with the ARB edges unable to exchange subunits, then the two edges of each ARB should move at the same rate. On the other hand, if the exchange is at the ARB edges, then, from data from microtubules in vitro, the poleward edge should move much faster than the kinetochore-ward edge. We found that the two edges of the ARB move at the same rate about half the time, but half the time they do not. Second, we studied the behaviour of two ARBs on a single fibre. If ARB motion is due solely to subunit addition at the kinetochore and loss at the pole, then the two ARBs must move poleward together. We found that after two ARBs are formed on a single fibre the region between the ARBs is unstable and rapidly depolymerizes. These results do not fit either model and suggest that influences of kinetochores and poles or other factors need to be considered that are not duplicated in experiments on microtubules in vitro.
Book review
Authors: Cutler D. 
Published: April 2021

ABSTRACT
Protein targeting is currently in a very interesting phase. For most of the pathways by which proteins traffic around the cell we now have at least preliminary descriptions obtained by morphological and fractionation techniques. Furthermore, the use of molecular biology in investigating the structure and function of the transported protein by mutagenesis and expression is making some important contributions toward our understanding. As well as these, the use of in vitro assays for transport processes has begun to complement the power of yeast genetics and, together, these are leading to the isolation of components of the machinery controlling transport.
Protein Targeting, edited by K. F. Chater, N. J. Brewin, R. Casey, K. Roberts, T. M. A. Wilson and R. B. Flavell. The Company of Biologists Ltd (obtainable from The Biochemical Society, Colchester CO2 8HP) 1989. Pp. 253. £35, $65, hardback
Protein Targeting, edited by A. P. Pugsley. Academic Press Inc. New York, 1989. Pp. xi+279. $45, hardback
A myofibrillar protein of insect muscle related to vertebrate titin connects Z band and A band: purification and molecular characterization of invertebrate mini-titin
Authors: Nave Rüdiger , Weber Klaus 
Published: April 2021

ABSTRACT
We show that myofibrils of insect flight and leg muscle contain a doublet of polypeptides with apparent molecular weights of 700K (K=103Mr) (Hmp I) and 600K (Hmp H), respectively. In Locusta migratoria high ionic strength extraction solubilizes only Hmp II, which is readily purified in native form. It probably reflects a proteolytic derivative of the non-extractable Hmp I. On the basis of its viscosity radius and sedimentation coefficient, Hmp II has a molecular weight of 600K and seems to consist of a single polypeptide chain. The highly asymmetric structure of the molecule is confirmed by rotary shadowing. The flexible rods have a uniform diameter of 3-4 nm and an average length of 260 nm. Polyclonal antibodies show crossreactivity between Hmp II and its putative precursor Hmp I. We discuss the similarities and differences between the larger titin l/titin II of vertebrate sarcomeric muscle and the smaller Hmp l/Hmp II of invertebrate muscle and conclude that the latter may reflect a mini-titin. In line with the smaller length, immunoelectron microscopy locates the insect mini-titin to the I band and a very short portion of the A band only, while vertebrate titin is known to connect the Z band to the M band. Mini-titin has also been purified from several other insects including Drosophila. Immunofluorescence microscopy on frozen sections shows that mini-titin is present in the sarcomeric muscles of various species from different invertebrate phyla. These include Annelida, Nematomorpha, Plathelmintha, Nemertea and Nematoda like Ascaris lumbricoides and Caenorhabditis elegans. This wide-spread occurrence of invertebrate mini-titin is confirmed by immunoblotting experiments.
Changes in cyclic AMP receptor properties during adaptation in Dictyostelium discoideum
Authors: Ludérus M. E. E. , Spijkers M. J. , Driel R. Van 
Published: April 2021

ABSTRACT
In developing Dictyostelium discoideum cells, binding of cyclic AMP to the chemotactic receptor has been shown to oscillate. These oscillations represent cycles of activation, adaptation and deadaptation of the cyclic AMP receptor system.
We studied the molecular basis of these oscillatory changes in cyclic AMP receptor binding. We developed a rapid method of lysing cells during the course of the oscillations. This method guaranteed good preservation of ligand binding properties of the cyclic AMP receptor.
We found that oscillations in cyclic AMP binding resulted from changes in receptor affinity. The total number of receptors did not significantly change during oscillations. Our experiments also showed that both GTP and GDP abolished oscillations in receptor binding completely, presumably by acting via a G protein. The guanine nucleotides reduced the affinity of the receptor at all time-points of the oscillation cycle to the minimal, i.e. adapted, level.
We conclude that the cyclic process of activation, adaptation and de-adaptation in D. discoideum, at cyclic AMP receptor level, involves changes in receptor-G protein interaction. During adaptation, the affinity of the cyclic AMP receptor decreases and the receptor becomes insensitive to guanine nucleotides.
High-resolution surface views of human lymphocytes during capping of CD4 and HLA antigens as revealed by immunogold fracture-flip
Authors: Pavan Antonio , Mancini Patrizia , Lucania Giuseppe , Frati Luigi , Torrisi Maria Rosaria , Silva Pedro Pinto da 
Published: April 2021

ABSTRACT
The surface ultrastructure of lymphocytes during capping of two transmembrane proteins is shown. As seen by fracture-flip the plasma membranes of human lymphocytes are covered by a high density of surface particles. Incubation in 30 % glycerol leads to aggregation of these surface particles. Immunogold labelling shows that the transmembrane proteins bearing HLA class I and CD4 antigens are confined to the particle aggregates. These results indicate that surface particles revealed by fracture-flip represent surface protrusions of integral membrane proteins seen as intramembrane particles in freeze-fractured lymphocytes.
During capping HLA or CD4 antigens aggregate into progressively larger patches and, finally, into single caps. As revealed by fracture-flip the patches/caps are seen as clearly differentiated raised platforms that are clearly and sharply demarcated relative to contiguous areas of the surface. In nonpatched (non-capped) regions, the pattern of distribution and apparent density of surface particles remain unaltered. Immunogold labelling clearly demarcates patches and caps, and shows that virtually no antigen molecules remain dispersed over the non-patched (non-capped) regions. Estimates of the surface density of either HI.A or CD4 antigens over the capped areas point to high planar concentrations of the transmembrane proteins that bear these antigens.
The protein kinase c inhibitor H-7 activates human neutrophils: effect on shape, actin polymerization, fluid pinocytosis and locomotion
Authors: Keller H. U. , Niggli V. , Zimmermann A. , Portmann R. 
Published: April 2021

ABSTRACT
The present study demonstrates new properties of H-7. The protein kinase inhibitor H-7 is a potent activator of several neutrophil functions. Stimulation of initially spherical nonmotile neutrophils elicits vigorous shape changes within a few seconds, increases in cytoskeletal actin, altered F-actin distribution, increased adhesiveness and a relatively small increase in pinocytic activity. H-7 has also chemokinetic activities. Depending on the experimental condition, H-7 may elicit or inhibit neutrophil locomotion. It failed to induce chemotaxis. Thus, the response pattern elicited by H-7 is different from that of other leukocyte activators such as chemotactic peptides, PMA or diacylglycerols. The finding that H-7 can elicit shape changes, actin polymerization and pinocytosis suggests that these events can occur without activation of protein kinase C (PKC). PMA-induced shape changes and stimulation of pinocytosis were not inhibited by H-7.
Regulation by calcium of proliferation and morphology of normal human tracheobronchial epithelial cell cultures
Authors: Chopra D. P. , Sullivan J. K. , Reece-Kooyer S. 
Published: April 2021

ABSTRACT
Human tracheobronchial epithelial cells have been serially passaged in serum-free medium. This serumfree model was employed to investigate the effects of different concentrations of Ca2+ (0.1, 1.0 and 2.0 mM) on multiplication and morphology of the cells. The responses were analysed in terms of growth kinetics, histochemical and ultrastructural alterations. Culturing of the cells in high Ca2+ (1.0-2.0mM) medium stimulated cell multiplication characterized by increased colony forming efficiency, greater number of cells per colony and cell population doublings per day. Additionally, the high Ca2+ concentrations induced proliferation in cultures grown to confluency in low Ca2+, (0.1 mM) medium. Cells propagated in low Ca2+ medium consisted of relatively heterogeneous cell populations, with mosteells staining positive with periodic acid-Schiff (PAS) reagent. Ultrastructurally the cells exhibited secretory vesicles and microvilli on their surfaces, small desmosomes and intercellular interdigitation between cells and numerous large secretory vesicles in the cytoplasm. The cells grown in high Ca2+ medium acquired characteristics of a highly proliferative phenotype. The cultures consisted of closely packed, relatively homogeneous cells that did not stain with PAS reagent. Their characteristic features were: absence of surface secretory vesicles, reductions of microvilli and intercellular interdigitations, and increases in size and number of desmosomal junctions. The results show that low Ca2+ in the culture medium inhibits cell multiplication and favors the secretory cell phenotype, while high Ca2+ levels stimulate cell multiplication and inhibit the secretory cell phenotype.
Monensin does not consistently inhibit the spreading of normal or duchenne fibroblasts on glass
Authors: Brown Susan C. , Fisher Derek , Lucy Jack A. 
Published: April 2021

ABSTRACT
The spreading on glass of monensin-treated normal and Duchenne fibroblasts has been investigated with the intention of extending this approach to a study of the comparative spreading of these cells on differing substrata. Untreated normal and Duchenne fibroblasts varied considerably in their ability to spread on glass. The spreading properties of normal and DMD fibroblasts treated in four different ways were compared: (1) pre-incubated and plated without monensin; (2) pre-incubated with, but plated without monensin; (3) pre-incubated without, but plated with monensin; (4) pre-incubated and plated with monensin. The response to plating with monensin (and preincubation/plating with monensin) also varied from patient to patient, but no statistically significant differences in the degree of spreading between the four treatment groups were observed in pooled data for either normal or dystrophic fibroblasts. Our data thus do not substantiate the previous finding of Pizzey et al. (1984) that Duchenne fibroblasts spread less well than normal fibroblasts after pre-incubation or plating with monensin, and possible explanations for this are discussed. The observations made are, however, consistent with the recent report that dystrophin is effectively not expressed in fibroblasts, and with the idea that the abnormal behaviour of endomysial fibroblasts in Duchenne dystrophy is a secondary consequence of their proximity to degenerating muscle.
Endocytosis in elongating root cells of Lobelia Erinus
Authors: Samuels A. L. , Bisalputra T. 
Published: April 2021

ABSTRACT
Endocytosis was demonstrated in elongating cortical and epidermal root cells of Lobelia erinus using the apoplast marker lanthanum nitrate. Lanthanum treatment produced electron-dense deposits throughout the cell wall, as well as in coated and smooth vesicles, partially coated reticula, and multi-vesicular bodies. This labelling pattern was observed in root cells that had been ultrarapidly frozen on a copper mirror and freeze-substituted (cryofixation) or fixed by conventional transmission electron microscope (TEM) techniques. The amount of endocytosis occurring was measured by counting the number of vesicles μm -2 in root cells at various stages of development. Endocytosis occurred most in actively elongating cells, and least in mature cells, which were no longer elongating. The relationship between endocytosis and active cell wall secretion suggests that endocytosis may be acting to remove excess plasma membrane material added during exocytosis of secretory vesicles.
Identification of proteins associated with microtubule-organizing centres and filaments in the oral apparatus of the ciliate Paramecium tetraurelia
Authors: Keryer Guy , Iftode Francine , Bornens Michel 
Published: April 2021

ABSTRACT
In an effort to study the assembly of microtubules in ciliates we have isolated the oral apparatus of Paramecium tetraurelia, a major microtubule-organizing centre (MTOC) and identified several proteins localized in the fibrillar system associated with basal bodies. Using a monoclonal antibody raised against human centrosomes (CTR210) and a polyclonal antibody (OF1) directed against a 87×10-3Mr protein of the oral apparatus of Tetrahymena, we observed the same decoration of the oral apparatus of Paramecium, both in situ and after isolation. Ultrastructural localization further showed the presence of the antigens in the fibrillar network that forms a layer under almost all the buccal cavity in close apposition to the basal bodies. CTR210 recognized two sets of polypeptides of Mr72 and 80×103, whereas OF1 recognized a single polypeptide of 80×103. Only the 80×103Mr polypeptide was also decorated with the monoclonal antibody MPM-2, previously shown to decorate the oral apparatus of Paramecium and known to react with phosphorylated epitopes in a large variety of MTOCs. All the proteins identified with the three antibodies are insoluble at high ionic strength, display several isoforms and apparently belong to the same fibrillar material. The function of this material in the spatial organization, the structure, and activity of the MTOCs is discussed.
Segregation of the myogenic cell lineage in mouse muscle development
Authors: Jones Gareth E. , Murphy Susan J. , Watt Diana J. 
Published: April 2021

ABSTRACT
With increasing interest in the idea of therapeutic implantation of normal muscle precursor cells into muscle lacking the protein product of the dystrophin gene, it has become important to obtain enriched populations of myogenic cells from biopsied muscle sources. Myogenic cells for implantation are highly favoured as they are the only cells that will fuse readily with host muscle fibres into which they are implanted, thus carrying the introduced gene into the target fibre with the maximum of efficiency. Second, myogenic cells appear less immunogenic than those of a non-myogenic nature; and third, the use of mononuclear myogenic cells may permit the introduction of multiple copies of a deficient gene into the patient’s own cells.
From a mixed population of cells obtained by the enzymic disaggregation of neonatal murine muscle we have selected, utilising a modification of the panning technique, for a cell population rich in myogenic cells. Segregation was accomplished using Mab H28, an antibody to the mouse neuronal cell adhesion molecule (N-CAM), derived from mouse/rat hybridoma cells. Following incubation with Mab H28, disaggregated muscle was applied to the surface of a bacteriological grade dish previously coated with anti-rat immunoglobulin. Cells segregated into two populations; those bearing N-CAM, and hence labelled with Mab H28, were adherent to the dish, whereas those not expressing N-CAM remained in suspension.
Use of this technique, which involves minimal cell loss, resulted in the segregation of prefusion myogenic cells together with fibroblasts in the ‘nonadherent’ fraction, whereas cells in the adherent fraction consisted of a highly enriched population of actively dividing myogenic cells.
Proteins of the mammalian mitotic spindle: phosphorylation/dephosphorylation of MAP-4 during mitosis
Authors: Vandré Dale D. , Centonze Victoria E. , Peloquin John , Tombes Robert M. , Borisy Gary G. 
Published: April 2021

ABSTRACT
The phosphoprotein composition of isolated CHO spindles was analyzed using the MPM-1 and MPM-2 antibodies, which are reactive with a phosphorylated epitope enriched in mitotic cells and present on the centrosome, kinetochores, midbody and fibers of the mitotic spindle. Several high molecular weight phosphorylated spindle proteins were detected on immunoblots, including species of 410 ×103Mr, 350 ×103Mr, a 230–240×103Mr doublet, 210×103 Mr and 120×103Mr. The temporal and spatial distribution of the MPM-reactive phosphoproteins was determined by examining spindle structures isolated from cells at various stages of mitosis. The susceptibility of the staining pattern to extraction with salt, a procedure known to remove most microtubule-associated proteins (MAPs), was also examined.
The phosphorylated 210×103Mr species was identified as MAP-4 and localized to the spindle fibers using (1) a polyclonal antibody raised against this species, that reacted with known MAPs, and (2) established MAP-4 antibodies that reacted with the spindle 210 × 103Mr MPM-reactive proteins. The comparative immunoblot and immunofluorescence analysis establishes a cycle of phosphorylation/ dephosphorylation of MAP-4 upon entry and exit from mitosis. Regarding the other MPM-reactive proteins, comparative immunofluorescence staining and immunoblot analysis of isolated spindle samples before and after salt extraction indicate that they may be constituents of the centrosome, kinetochores or midbody, but their definitive identification awaits the production of monospecific antibodies..
Immunoelectron microscopic analysis of the intracellular distribution of primer recognition proteins, annexin 2 and phosphoglycerate kinase, in normal and transformed cells
Authors: Kumble Krishnanand D. , Vishwanatha Jamboor K. 
Published: April 2021

ABSTRACT
Primer recognition proteins (PRP) are accessory proteins for DNA polymerase a in lagging strand DNA replication. We have previously reported that the PRP consist of a complex of two proteins identified as 3-phosphoglycerate kinase (PGK) and the protein-tyrosine kinase substrate, annexin 2 monomer. The physiological role of annexin 2 is not known. Two pools of annexin 2 exist in cells. A majority of annexin 2 is localized with the plasma membrane as a heterotetramer in association with a light chain. Monomer annexin 2 is cytosolic. The identification of annexin 2 monomer as a part of the PRP complex represents one of the physiological roles of this protein in cells. To function as PRP, annexin 2 and PGK would have to be present in the cell nucleus. To investigate whether monomer annexin 2 is indeed associated with nuclear DNA synthesis, we investigated the presence of annexin 2 and PGK in the cell nucleus. In this paper, we demonstrate the presence of annexin 2 and PGK in nuclear extracts. The nuclear fraction of these proteins represents a small subset of the total cellular pools. Immunoelectron-microscopic analyses using anti-PRP antisera demonstrate the distribution of these proteins in HeLa cell nuclei and cytoplasm. Under identical conditions, an anti-cyto-keratin monoclonal antibody preferentially labels the plasma membrane without detectable intracellular staining. The distribution of annexin 2 and PGK in both nuclei and cytoplasm is similarly observed in cells from normal tissues such as freshly isolated rat hepatocytes and hamster pancreatic tissue. The results presented in this report further substantiate the involvement of annexin 2 and PGK, as part of the PRP complex, in nuclear DNA synthesis.