bluuebunny/crossref_metadata_2025_split huggingface dataset:
Scanning force microscopy of microtubules and polymorphic tubulin assemblies in air and in liquid
Authors: Vater W. , Fritzsche W. , Schaper A. , Böhm K. J. , Unger E. , Jovin T. M. 
Published: April 2021

ABSTRACT
We have investigated microtubules (MTs) and polymor-phic assemblies, formed in vitro from isolated microtubule protein, by scanning force microscopy (SFM) in air and in liquid. Immobilization of MTs was achieved by placing a drop of the assembly solution on a polylysine-coated coverslip. After washing with taxol and air drying, the characteristic microtubular fibrous morphology appeared in the SFM. The MTs formed a network similar to that obtained by transmission electron microscopy (TEM). A height of ∼9.5 nm for dried MTs was computed from the surface topography. Glutaraldehyde fixation of the MTs yielded higher structures (∼14 nm), which swelled to ∼20 nm after rehydration, a value close to the MT diameter of ∼25 nm determined from TEM images of ultrathin sections. The protofilament pattern of the MTs and surface attached MT-associated proteins were not apparent from SFM, although the height along the long axis of the MTs appeared slightly modulated. In addition to MTs, various polymorphic tubulin assemblies including ribbons, hoops and double-walled MTs were visualized by SFM.
High molecular weight polypeptides related to dynein heavy chains in Nicotiana tabacum pollen tubes
Authors: Moscatelli Alessandra , Casino Cecilia Del , Lozzi Luisa , Cai Giampiero , Scali Monica , Tiezzi Antonio , Cresti Mauro 
Published: April 2021

ABSTRACT
Nicotiana tabacum pollen tubes contain two high molecular weight polypeptides (about 400 kDa), which are specifically expressed during pollen germination and pollen tube growth in BK medium. The high molecular weight doublet resembles the dynein heavy chains in some biochemical properties. Sedimentation profiles of pollen tube extracts show that the high molecular weight bands have sedimentation coefficients of 22 S and 12 S, respectively. ATPase assay of sedimentation fractions shows an activity ten times higher when stimulated by the presence of bovine brain microtubules in fractions containing the 22 S high molecular weight polypeptide. Both these high molecular weight polypeptides can bind microtubules in an ATP-dependent fashion. A mouse antiserum to a synthetic peptide reproducing the sequence of the most conserved ATP-binding site among dynein heavy chains recognized the two high molecular weight polypeptides. Therefore these polypeptides have sequences immunologically related to the ATP binding sites of dynein heavy chains.
The functional thrombin receptor is associated with the plasmalemma and a large endosomal network in cultured human umbilical vein endothelial cells
Authors: Horvat R. , Palade G. E. 
Published: April 2021

ABSTRACT
The functional thrombin receptor, normally expressed by endothelial cells and platelets, is a member of the G protein-coupled, seven membrane-spanning-domain receptor family and is thought to be responsible for most, if not all, the cell stimulatory effects of thrombin. Upon binding, thrombin cleaves the receptor’s N-terminal ectodomain, unmasking a new N terminus, which by itself activates the receptor. Using antibodies to different domains of the human thrombin receptor, we have localized the receptor in cultured human umbilical vein endothelial cells by indirect immunofluorescence and immunoelectron microscopy. We found the receptor expressed on the plas-malemma of cultured endothelial cells in individual units rather than in clusters, at lower concentration than, and at different sites from, thrombomodulin. We also found the receptor associated with a distinct, intracellular, transfer-rin receptor-containing, tubulovesicular network. The thrombin receptor-positive structure spread from the per-inuclear region to the periphery of the cells, exhibiting a number of varicosities interconnected by branching tubular elements, strikingly similar to an image recently described for a continuous endosomal reticulum. Our results provide morphological evidence for the presence of the functional thrombin receptor at relative low density on the surface of cultured endothelial cells (compared to thrombomodulin) and in relatively large quantities inside the cells, associated with an endosomal compartment.
A high molecular mass protein isolated from chicken gizzard: its localization at the dense plaques and dense bodies of smooth muscle and the Z-disks of skeletal muscle
Authors: Terasaki Asako Goi , Nakagawa Hiroyuki , Kotani Eiji , Mori Hajime , Ohashi Kazuyo 
Published: April 2021

ABSTRACT
We purified a 450 kDa protein from a low-salt alkaline extract of chicken gizzard smooth muscle. This high molecular mass protein could be extracted with the low-salt alkaline solution at 37°C but not at 4°C. The 450 kDa protein was isolated from the extract by ammonium sulfate fractionation and following sequential column chromatography using hydroxylapatite, DEAE-Cellulofine A-800m and phenyl-Sepharose CL-4B resins. The partially purified protein molecule resembled a flexible rod with a globular head and an irregular-shaped tail. Its length was approxi-mately 300 nm. The nucleotide sequence of the partial cDNA encoding this protein was determined and analyzed with a data base. The analysis showed that the protein revealed significant homology with the rod region of chicken filamin (57% homology in amino acid sequence). Immunoblot analysis showed that an affinity-purified antibody reacted exclusively with the 450 kDa protein band of smooth, skeletal and cardiac muscle tissues. By indirect immunofluorescence microscopy, we examined the local-ization of the 450 kDa protein in smooth and skeletal muscle cells. The affinity-purified antibody against the 450 kDa protein stained the dense plaques and dense bodies of smooth muscle, the peripheral region of Z-disks and the subsarcolemmal region of skeletal muscle. Immunoelectron microscopy confirmed the localization of the 450 kDa protein at the peripheral regions of the actin anchoring structures mentioned above. Judging from its amino acid sequence, molecular size, molecular shape, immunological reactivity and localization in muscle cells, the 450 kDa protein seemed to be a new component associated with the actin-anchoring structures of muscle tissues. We tenta-tively call this protein ‘fulcin’.
A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication
Authors: Starborg Maria , Brundell Eva , Gell Katarina , Larsson Catharina , White Irene , Daneholt Bertil , Höög Christer 
Published: April 2021

ABSTRACT
We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3-5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromo-somal localization of the P1 gene was determined by fluo-rescence in situ hybridization to region 6p12 in human metaphase chromosomes.
Phagocytosis of apoptotic bodies by liver endothelial cells
Authors: Dini Luciana , Lentini Alessandro , Diez Guzman Diez , Rocha Marian , Falasca Laura , Serafino Lucia , Vidal-Vanaclocha Fernando 
Published: April 2021

ABSTRACT
Using electron microscopy and cytofluorimetry we studied the role of carbohydrate-specific recognition systems in the interaction of apoptotic bodies with normal and interleukin 1-activated sinusoidal endothelial cells. Microfluorimetric observation of liver tissue sections revealed octadecyl-rhodamine B-labelled apoptotic body binding to the sinu-soidal wall of mouse liver, when they were injected intra-portally. Plate-scanning cytofluorimetry demonstrated that about 20-25% of Acridine Orange-labelled apoptotic bodies could adhere specifically to cultured endothelial cells after 15 minutes of incubation. Adhesion increased to 30% when the cells were incubated for 60 minutes. Using a mixture of galactose/N-acetylglucosamine/mannose as competition solution apoptotic body adhesion was signifi-cantly reduced especially after longer times of incubation, when the percentage of inhibition reached 50%. Following 4 hours exposure of liver endothelial cells to 1 ng/ml human recombinant interleukin-1β adhesion markedly increased after 60 minutes of incubation, whereas the co-incubation of interleukin-1β with the inhibitors brings down the adhesion to basal values obtained in controls. Electron microscopic observation of the adhesion process showed that the number of endothelial cells binding apoptotic bodies gradually increased from low to high values with time. After 60 minutes of incubation, the majority of apoptotic bodies were seen inside phagosomes and only a few remained at the cell surface. Liver endothelial cells bound and endocytosed apoptotic bodies through carbohy-drate-specific receptors. Moreover, this scavenger action was interleukin-1 enhanced, thus suggesting its possible activation during inflammatory and immune processes.
αv and α3 integrin subunits are associated with myofibrils during myofibrillogenesis
Authors: McDonald K. A. , Lakonishok M. , Horwitz A. F. 
Published: April 2021

ABSTRACT
The development of the myofibrillar apparatus in skeletal muscle is a process in which transmembrane linkages with adhesion molecules are implicated. Integrins are one class of transmembrane adhesion receptors which appear to mediate these interactions. Two prominent linkages are at the myotendinous junction (MTJ), which resides at the ends of the cell and connects myofibrils to the tendon, and the costameres, which encircle the girth of the cell and connect the Z-disks to the sarcolemma. In this study we report that the αv integrin subunit is a prominent component of the costamere. The αv subunit is present initially on developing myotubes in a diffuse staining pattern with some concentration along nascent myofibrils. However, it appears in a striated pattern at the costamere and inconsistently at the M-line following the striation of α-actinin and titin but before that of desmin. Its recruitment to preformed striation suggests that it is incorporated into a pre-existing structure. The presence of αv in the costamere points to a role in lateral myofibrillar anchorage. In addition, we find that the α3 subunit is transiently associated with myofibrils along portions of their lengths and at their ends during myofibrillogenesis. The α3 subunit staining shows a novel localization and junctional structure. As myofibrils become striated the α3 integrin dis-sociates from the localized pattern and becomes diffuse. This suggests a possible role in the stabilization of nascent myofibrils prior to striation. Antibody-induced perturba-tion of adhesion mediated by the integrin β1 subunit in developing myotubes inhibits assembly of the sarcomeric architecture. The observations presented here also con-tribute to an emerging theme, where different integrins occupy unique junctional regions on muscle.
Neutralisation of TGF-β1 and TGF-β2 or exogenous addition of TGF-β3 to cutaneous rat wounds reduces scarring
Authors: Shah Mamta , Foreman David M. , Ferguson Mark W. J. 
Published: April 2021

ABSTRACT
Exogenous addition of neutralising antibody to transforming growth factor-β1,2 to cutaneous wounds in adult rodents reduces scarring. Three isoforms of transforming growth factor-β (1, 2 and 3) have been identified in mammals. We investigated the isoform/isoforms of TGF-β responsible for cutaneous scarring by: (i) reducing specific endogenous TGF-β isoforms by exogenous injection of isoform specific neutralising antibodies; and (ii) increasing the level of specific TGF-β isoforms by exogenous infiltration into the wound margins. Exogenous addition of neutralising antibody to TGF-β1 plus neutralising antibody to TGF-β2 reduced the monocyte and macrophage profile, neovascu-larisation, fibronectin, collagen III and collagen I deposi-tion in the early stages of wound healing compared to control wounds. Treatment with neutralising antibodies to TGF-βs 1 and 2 markedly improved the architecture of the neodermis to resemble that of normal dermis and reduced scarring while the control wounds healed with scar formation. Exogenous addition of neutralising antibody to TGF-β1 alone also reduced the monocyte and macrophage profile, fibronectin, collagen III and collagen I deposition compared to control wounds. However, treatment with neutralising antibody to TGF-β1 alone only marginally reduced scarring. By contrast, wounds treated with neu-tralising antibody to TGF-β2 alone did not differ from control wounds. Interestingly, exogenous addition of the TGF-β3 peptide also reduced the monocyte and macrophage profile, fibronectin, collagen I and collagen III deposition in the early stages of wound healing and markedly improved the architecture of the neodermis and reduced scarring. By contrast, wounds treated with either TGF-β1 or with TGF-β2 had more extracellular matrix deposition in the early stages of wound healing but did not differ from control wounds in the final quality of scarring. This study clearly demonstrates isoform specific differ-ences in the role of TGF-βs in wound healing and cutaneous scarring. TGF-β1 and TGF-β2 are implicated in cutaneous scarring. This study also suggests a novel therapeutic use of exogenous recombinant, TGF-β3 as an anti-scarring agent.
Heterochromatin protein 1 distribution during development and during the cell cycle in Drosophila embryos
Authors: Kellum Rebecca , Raff Jordan W. , Alberts Bruce M. 
Published: April 2021

ABSTRACT
Heterochromatin protein 1 (HP1) was initially discovered as a protein that is associated with the heterochromatin at the chromocenter of polytene chromosomes in Drosophila larval salivary glands. In this paper we investigate the localization of heterochromatin protein 1 in the diploid nuclei of Drosophila embryos. We focus on its association with the interphase heterochromatin in fixed embryos before and during cycle 14, the developmental time at which heterochromatin becomes most conspicuous, and also follow its localization during mitosis. The GAGA transcription factor was recently shown to be localized at sequences within α-heterochromatin in pre-cycle 14 embryos, and an antibody against this protein serves as a convenient marker for these sequences.
We find an enrichment of heterochromatin protein 1 in the intensely DAPI-staining regions near the apical surface of nuclear cycle 10 embryos. At this stage GAGA factor is localized into punctate structures in this same region. This enrichment for HP1 is markedly increased during nuclear cycle 14.
Surprisingly, whereas GAGA factor retains its association with the heterochromatin throughout the cell cycle, a significant fraction of HP1 is dispersed throughout the spindle around the segregating chromosomes during mitosis. This dispersed pool of heterochromatin protein 1 was observed during mitosis in both early and late Drosophila embryos and in an analysis of a bacterially produced 6× histidine-heterochromatin protein 1 fusion protein injected into living Drosophila embryos. When Drosophila tissue culture cells were prepared by a method which removes soluble protein and avoids fixation of the mitotic chromosomes, an enrichment for heterochromatin protein 1 in the heterochromatin of the chromosomes was discovered also.
The expression of megalin (gp330) and LRP diverges during F9 cell differentiation
Authors: Czekay Ralf-Peter , Orlando Robert A. , Woodward Luann , Adamson Eileen D. , Farquhar Marilyn G. 
Published: April 2021

ABSTRACT
The receptor-associated protein, RAP, is a chaperonin-like molecule that binds to two members of the low density lipoprotein receptor (LDLR) superfamily – megalin (gp330) and the LDL receptor-related protein (LRP). In F9 embryonal carcinoma cells, expression of RAP mRNA increases when differentiation is induced with retinoic acid and dibutyryl-cyclic AMP. We have investigated the expression of megalin and LRP and their interaction with RAP in F9 cells using biochemical and immunocytochemical methods. Both receptors are expressed in uninduced F9 cells, but only megalin co-precipitates with RAP. When F9 cells were induced to differentiate into parietal endoderm, the expression of megalin was dramatically increased. The expression of megalin exceeded that of LRP and RAP by an order of magnitude and both receptors co-precipitated with RAP. By immunoelectron microscopy, megalin and LRP were localized to clathrin-coated pits at the cell surface in both undifferentiated and differentiated F9 cells, whereas RAP was found mainly in the ER. A sizeable pool of LRP was also detected in the ER. When F9 cells were grown in suspension in the presence of RA and induced to develop into embryoid bodies, the expression of megalin and LRP segregated into different cell types: megalin was found in the outer epithelial layer and LRP in the stem cells of the inner core. Our results demonstrate that F9 cells induced to differentiate in monolayer culture express megalin, LRP and RAP, and RAP is capable of interacting simultaneously with both receptors. In embryoid bodies the expression of megalin and LRP diverges, and only megalin is expressed in the outer epithelial layer. The high levels of expression and cell specificity of megalin and LRP suggest important roles for these receptors in early embryonic development.
Import of microinjected proteins bearing the SKL peroxisomal targeting sequence into the peroxisomes of a human fibroblast cell line: evidence that virtually all peroxisomes are import-competent
Authors: Hill Paula E. , Walton Paul A. 
Published: April 2021

ABSTRACT
Peroxisomes import virtually all of their membrane and matrix proteins post-translationally. It is presently unknown whether, in mammalian cells, their exists a pool of mature peroxisomes which have received their complement of proteins and are import-incompetent. Previous work has shown that fibroblasts are capable of importing microinjected peroxisomal proteins into peroxisomes. This report describes the import of a hybrid peroxisomal protein into virtually all peroxisomes of the microinjected cell. The peroxisomal import was uniform in both short and long incubations. Pretreatment of the cells with cycloheximide did not affect the import of the peroxisomal protein, nor was there any difference in the distribution of the imported protein. Sequential microinjection experiments demonstrated that peroxisomes that had imported luciferase were capable of importing another peroxisomal protein injected 24 hours later. These results suggest that, in fibroblasts, all peroxisomes have associated protein-import machinery; this evidence does not support the hypothesis that there exists a pool of import-incompetent peroxisomes.
Cytoplasmic chaperonin complexes enter neurites developing in vitro and differ in subunit composition within single cells
Authors: Roobol Anne , Holmes Fiona E. , Hayes Nandini V. L. , Baines Anthony J. , Carden Martin J. 
Published: April 2021

ABSTRACT
Chaperonins containing t-complex polypeptide-1 (CCT) are cytosolic molecular chaperone particles implicated especially in the biogenesis of cytoskeletal proteins by promoting the correct folding of the major ubiquitous cytoskeletal components, tubulin and actin. We have purified cytosolic chaperonins from the ND7/23 cell line, determined their subunit composition and examined changes in the intracellular locations of their components during differentiation of ND7/23 cells to a neuronal phenotype by using immunocytochemistry and immunoblots. Chaperonins containing the CCTα (TCP1? subunit enter neuritic processes and are particularly noticeable at the leading edge of growth cone-like structures where they co-localise with actin. Chaperonins containing three other components (CCTβ, ε and γ), however, remain predominantly restricted to perikaryal cytoplasm. These findings suggest a heterogeneous population of chaperonin particles within single differentiated ND7/23 cells and this may reflect specialisation of chaperonin function in different cytoplasmic compartments of a neurone. Further, since ribosomes do not enter neurites while CCTα-containing chaperonins do, the latter may play roles, subsequent to translation, which influence cytoskeletal elaboration during neuritogenesis.
Stationary organization of the actin cytoskeleton in Vallisneria: the role of stable microfilaments at the end walls
Authors: Ryu Jung-Hwa , Takagi Shingo , Nagai Reiko 
Published: April 2021

ABSTRACT
In mesophyll cells of the aquatic angiosperm Vallisneria gigantea, bundles of microfilaments (MFs) serve as tracks for the rotational streaming of the cytoplasm, which occurs along the two longer side walls and the two shorter end walls. The stationary organization of these bundles has been shown to depend on the association of the bundles with the plasma membrane at the end walls. To identify the sites of such association, the effects of cytochalasin B (CB) on the configuration of the bundles of MFs were examined. In the case of the side walls, MFs were completely disrupted after treatment with CB at 100 μg/ml for 24 hours. By contrast, in the case of the end walls, a number of partially disrupted MFs remained even after 48 hours of treatment. After removal of CB, a completely normal arrangement of bundles of MFs was once again evident within 24 hours after a rather complicated process of reassembly. When reassembly had been completed, the direction of cytoplasmic streaming was reversed only in a small fraction of the treated cells, suggesting that bundles of MFs are anchored and stabilized at the end walls of each cell and that the polarity of reorganized bundles and, therefore, the direction of the cytoplasmic streaming is determined in a manner that depends on the original polarity of MFs that remained in spite of the disruptive action of CB. By contrast, the direction of reinitiated cytoplasmic streaming was reversed in 50% of cells in which the bundles of MFs had been completely disrupted by exogenously applied trypsin prior treatment with CB. The results confirm that protease-sensitive anchoring of microfilament bundles at the end walls is crucial for maintenance of the unique, stationary organization of the tracks for cytoplasmic streaming in these cells.