bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitation of actin polymerization in two human fibroblast sub-types responding to mechanical stretching
Authors: Pender Neil , Mcculloch Christopher A. G. 
Published: April 2021

ABSTRACT
To study early reorganization of the cytoskeleton in response to physical forces, human gingival and periodontal ligament fibroblasts were cultured on flexible plastic substrata and stretched by mechanical deformation of the substratum. F-actin was measured by quantitative spectrofluorimetry of FITC-phalloidin-stained cells. Fluorescence due to FITC-phalloidin was reduced stoichiometrically by co-incubation with unlabeled phalloidn. Increases in stretch-induced fluorescence were blocked by preincubation of cells with cytochalasin D. Compared to baseline values, F-actin in gingival fibroblasts was reduced by 50% at 10 s after stretching but was increased more than 100% at 50 s after stretching. Increases of F-actin were also dependent on the amount of deformation of membrane: 3.3% stretching produced the largest increase and 9.2% stretching produced the least. Stretch-activated actin polymerization was blocked by pre-incubation of cells with 500 ng ml−1 pertussis toxin or 10 mM EGTA. Compared to gingival cells, periodontal ligament fibroblasts exhibited twice the total actin per cell and three times the baseline F-actin per mm2, however, there was no increase in fluorescence after stretching. In both cell types, stretching caused increased ruffling activity and no detectable shape changes at 60 s, but at 180 s the cells shortened and pseudopods retracted. The microfilament system of fibroblasts exhibits rapid dynamic responses to mechanical deformation prior to cell shape changes and appears to be dependent on the cell type.
Evidence for an inequality in the forces that generate principal and reverse bends in sperm flagella
Authors: Eshel Dan , Shingyoji Chikako , Yoshimura Kenjiro , Gibbons Ian R. , Takahashi Keiichi 
Published: April 2021

ABSTRACT
The response of the mechanism initiating flagellar bends to imposed mechanical transients has been studied by holding the head of a sea urchin sperm in the tip of a sinusoidally vibrating micropipet and then displacing the micropipet laterally at a speed of up to 1.15 μm ms−1 for 1.5 beat cycle, without vibration, before resuming sinusoidal vibration with the initial phase, frequency and amplitude at the new location of the pipet This transient displacement of the micropipet delays the initiation of the bend that was due to initiate 0.5 beat cycle after onset of the displacement. The amount of this delay increases with the speed of the displacement, for speeds up to 1 μm ms−1. Analysis of the flagellar waveforms during the transient showed that with imposed displacements at speeds of equal magnitude, the initiation of a principal bend was delayed to a longer extent thanthat of a reverse bend. At a micropipet speed of 0.75 μm ms−1, there was an average delay of 0.21 beat cycle in the initiation time of a principal bend as compared to a delay of only about 0.04 beat cycle in the initiation time of a reverse bend during displacements in the opposite direction. For both principal and reverse bends, the second bend due to initiate during the transient displacement initiated in most of the cases with no delay, regardless of the micropipet speed. Our results suggest that the force generated by microtubule sliding to initiate a new reverse bend is greater than that generated to initiate a principal bend.
Synchronised secretory organelle docking in Paramecium saltatory movement along microtubules transiently formed from ciliary basal bodies and selective exclusion of microinjected heterologous organelles
Authors: Glas-Albrecht René , Kaesberg Birgit , Knoll Gerd , Allmann Karl , Pape Regina , Plattner Helmut 
Published: April 2021

ABSTRACT
Ln certain strains (nd) of Paramecium tetraurelia all secretory organelles (trichocysts) can be detached from the cell surface and then reattached synchronously. To account for the lability of microtubules involved in trichocyst docking during the preparation procedures we analysed this process by combining video microscopy, analogue contrast enhancement microscopy, laser scanning fluorescence microscopy (both after fast freezing, freeze-substitution and anti-tubulin antibody fluorescence labelling), and electron microscopy after cryoflxation. We found that synchronous trichocyst docking is saltatory, occurring along transiently formed microtubules (not normally recognised in these cells), which emanate from ciliary basal bodies, acting as organising centres. Hence, trichocyst transport proceeds from the plus to the minus end of microtubules (in contrast to gland cells), with the ‘correct’ polarity (trichocyst tips pointing to the cell surface). We then injected chromaffin granules (isolated from bovine adrenal medullae) during the trichocyst detachment phase and analysed cells by electron microscopy during and after synchronised redocking of trichocysts. We used a chromate reaction for chromaffin granule identification on semithin sections by X-ray microanalysis (scanning transmission EM). While chromaffin granules remained intact (as judged by morphology and Cr signals) and although cell function was unimpaired (as judged by complete trichocyst attachment), we determined that heterologous organelle transport was not detectable, probably because of inverse microtubule polarity.
Multiple families of proteins in the secretory granules ofParamecium tetraurelia:Immunological characterization and immunocytochemical localization of trichocyst proteins
Authors: Shih Shian-Jiun , Nelson David L. 
Published: April 2021

ABSTRACTParamecium tetraurelia has thousands of secretory granules (trichocysts), which release their protein contents by regulated exocytosis. The secretory proteins that fill the granule comprise the condensed trichocyst matrix (ctmx), a paracrystalline structure that, upon exocytosis, expands about eightfold in length within milliseconds. The resulting needle-like extended trichocyst matrix (xtmx), also paracrystalline, is released outside the cell. Both ctmx and xtmx are composed of 35 or more small (Mr 14–25×103), acidic (pI4.4–5.8) proteins. We used monoclonal antibodies (mAbs) raised against proteins of the xtmx to study the relationship among these proteins, and to determine their locations within the paracrystalline ctmx and xtmx.The antibodies defined four distinct protein groups. Group I proteins (defined by mAb Al-3 and B5–5) showed a relatively wide range of pl values, and existed in xtmx as disulfide-linked heterodimers. They were distributed throughout the matrix of condensed and extended trichocysts, as judged by electron-microscopic immunocytochemistry. Group n proteins (defined by mAb B4–4 and B3–5) were more acidic, also present as heterodimers and specifically localized in a 150 nm wide cortex in ctmx and in a much thinner cortex in xtmx. In xtmx, antibodies against group II proteins stained the outer surface on the regions between the electrondense striations with 55 nm intervals. However, these regions were not accessible to antibody B4–4 in ctmx. Group IH proteins (defined by mAb B7–4) are monomeric proteins; group IV are two subunits of heterodimers. Proteins of groups IH and IV were localized in the core of ctmx, but were distributed uniformly in xtmx.Our results show that these very similar tmx proteins are not structurally equivalent. Within the highly regular structures of condensed and extended tmx, immunologically distinct families of tmx proteins occupy specific and different positions in the paracrystalline array. One family of tmx proteins (group II) is buried in the condensed tmx and only becomes accessible to antibodies upon trichocyst extension. Our results suggest that the 150 nm cortex of condensed tmx expands lengthwise, while decreasing in the thickness, to form the outer shell of extended tmx, and the core expands in length without decreasing in diameter to form the inside structure of the extended tmx.
A Drosophila anti-rna polymerase ii antibody recognizes a plant nucleolar antigen, rna polymerase i, which is mostly localized in fibrillar centres
Authors: Martin Marta , Medina Francisco Javier 
Published: April 2021

ABSTRACT
The distribution of nucleolar RNA polymerase in the nucleolus of onion root meristematic cells has been studied by means of an antibody originally raised against Drosophila RNA polymerase II. This antibody recognizes the homologous domains of the large subunit of the enzyme, which are highly conserved throughout evolution in the three classes of eucaryotic RNA polymerases. Given that RNA polymerase I is confined to the nucleolus, and that the onion cell nucleolus lacks digitations of extranuc-leolar chromatin, we conclude that the nucleolar enzyme localized is RNA polymerase I. A quantitative approach, independent of the existence of borderlines between nucleolar fibrillar centres and the dense fibrillar component, allowed us to show that the enzyme is localized in fibrillar centres and in the transition area between them and the dense fibrillar component, in parallel with the nucleolar DNA.
These results, together with previous autoradiographic, cytochemical and immunocytochemical results, in this and other species, lead us to conclude that the activation of rDNA for transcription occurs in the fibrillar centres and pre-rRNA synthesis is expressed at the transition area between fibrillar centres and the dense fibrillar component. Fibrillar centres are connected to each other by extended RNA polymerase-bound DNA fibres, presumably active in transcription. This work provides evidence of the high evolutionary conservation of some domains of the large subunit of RNA polymerases and of the existence of fibrillar centres in the nucleolus of plant cells, totally homologous to those described in mammalian cells.
Visualization of centromeric and nucleolar DNA in fission yeast by fluorescence in situ hybridization
Authors: Uzawa Satoru , Yanagida Mitsuhiro 
Published: April 2021

ABSTRACT
The nucleolar and centromeric DNAs of the fission yeast Schizosaccharomyces pombe were visualized in the nucleus by fluorescence in situ hybridization using repetitive ribosomal and centromeric DNAs as the probes. The rDNAs were seen in the nuclear domain previously assigned as nucleolar, that is, the region into which the rod-like chromatin protrudes from the hemispherical chromosomal domain. Using mitotically-arrested cells containing condensed chromosomes, it was demonstrated that the rDNAs were present on the smallest chromosome HI, consistent with genetic data. Using a centromeric repetitive element as the hybridization probe, the centromere of chromosome HI, cen3, which contains the largest number of the repetitive elements, was visualized. The centromere in interphase cells is located near the periphery of the nucleus as a single dot. Early in mitosis, however, it divides into two and is situated in the middle of the short mitotic spindle. After spindle extension in anaphase, the centromeric DNA is present at both ends of the spindle, that is, near the spindle pole bodies. The movement of cen3 during mitosis (anaphase A and B) is discussed in relation to spindle dynamics and chromosome separation.
Protein kinase C involvement in focal adhesion formation
Authors: Woods Anne , Couchman John R. 
Published: April 2021

ABSTRACT
Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form.
Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h and then treated with kinase inhibitors H7 and HA1004 for 2h, IRM indicated a reduction in focal adhesion formation at concen trations where protein kinase C (PKC) should be inhibited. In contrast, focal adhesions formed normally at concentrations of these inhibitors where cyclic AMP- or cyclic GMP-dependent kinases should be inactivated. Inhibition of PKC, but not that of cyclic AMP- or cyclic GMP-dependent kinases, also prevented the formation of stress fibers and induced a dispersal of talin and vinculin, but not integrin β1 subunits, from small condensations present at 1h. Consistent with the reduction in focal adhesion formation when PKC was inhibited, activation of PKC by 30 minutes of treatment with phorbol esters induced focal adhesion formation in cells spread for 3h on substrata composed of the cellbinding (RGD-containing) fragment of fibronectin, while untreated cells or those treated with inactive phorbol esters did not form these structures.
Characterization of endoplasmic reticulum by co-localization of BiP and dicarbocyanine dyes
Authors: Terasaki Mark , Reese Thomas S. 
Published: April 2021

ABSTRACT
The original concept of endoplasmic reticulum derived from the observation of a reticular network in cultured fibroblasts by electron microscopy of whole cells. It was previously reported that the fluorescent dye, DiOC6(3), stains a similar network as well as mitochondria and other organelles in living cells. Here, we investigate the significance of the structures labeled by DiOC6(3) in CV1 cells, a monkey epithelial cell line. First, we show that the network stained in living CV-1 cells is preserved by glutaraldehyde fixation and then we co-label it with an antibody against BiP (immunoglobulin binding protein), a protein commonly accepted to be present in the endoplasmic reticulum. Anti-BiP labeled the same network as that labeled by DiOC6(3), so this network now is identified as being part of the endoplasmic reticulum. DiOC6(3) labels many other membrane compartments in addition to the endoplasmic reticulum. This, along with its lipophilic properties, suggests that DiOC6(3) stains all intracellular membranes. However, the extensive reticular network in the thin peripheral regions of cultured cells is easily distinguished from these other membranes. Thus, staining by DiOC6(3) is a useful method for localizing the endoplasmic reticulum, particularly in thin peripheral regions of cultured cells.
Diacylglycerol activation of protein kinase C results in a dual effect on Na+,K+-ATPase activity from intact renal proximal tubule cells
Authors: Bertorello Alejandro M. 
Published: April 2021

ABSTRACT
This study evaluated the effect of L-1-oleoyl-2-acetoyl sn-3-glycerol (OAG) on ouabain-sensitive Na,K-depen dent oxygen consumption (Na,K-QO2) in intact renal proximal tubule cells (RPTC). Basal Na,K-QO2 (nmol O2/mg protein per min) was 20.0±1.0. Incubation with 10 nM of OAG induced a dual effect on Na,K-QO2, with an initial stimulation (maximal at 10 min, 37.1±5.0), followed by an inhibition (significant at 20 min, 16.3±1.0). No changes in ouabain-insensitive QO2 were observed in any of the protocols. The effects were abolished by sphingosine, a protein kinase C inhibitor. Stimulation was abolished by amHorlde 0.1 mM. Amiloride had no effect on Na,K-QO2 at the concen tration used. Stimulation was not potentiated by the sodium ionophore, amphotericine B, and the later inhibition was still observed in the presence of ampho tericine B. The initial stimulation was attributed to an increase in sodium permeability, while the later inhi bition was attributed to a direct effect on the Na,K pump. Regulation of Na+,K+-ATPase activity by pro tein kinase C in intact RPTC can be accomplished by a direct effect on the protein or as a secondary effect consequent upon changes in intracellular sodium.
Attachment of steroidogenic lipid droplets to intermediate filaments in adrenal cells
Authors: Almahbobi Ghanim , Williams Lindy J. , Hall Peter F. 
Published: April 2021

ABSTRACT
Light microscopy of living and extracted adrenal cells (Y-l mouse adrenal tumour cells and cultured bovine fasciculata cells), using Nomarski optics and fluorescence with nile red to stain lipid, revealed in both cell types that lipid droplets remain attached to intermediate filaments when the cells are extracted to prepare these structures.
Electron microscopy of thin sections shows the presence of lipid droplets in both cell types. The droplets differ in appearance but are, in both cases, surrounded by a complete capsule 5nm wide. The droplets in Y-l cells include those associated with lysosomes and crystalline structures in addition to typical rounded forms. Only the latter type is seen in bovine fasciculata. Intermediate filaments apparently ending in droplets can also be seen. Immunoelectron microscopy with anti-vimentin and Protein A conjugated to gold particles together with measurement of the diameter of these structures identifies them as intermediate filaments. When adrenal cells are permeabilised and extracted under mild or severe conditions using Triton X-100, thin sections showed that lipid droplets remain associated with the cytoskeleton and in particular intermediate filaments. Extraction under mild and severe conditions cleared the cell contents, revealing attachment of intermediate filaments to lipid droplets with greater clarity than in unextracted cells, i.e. homogenised cells or cells subjected to lysis. Such attachment was unequivocally demonstrated in stereo pairs. These observations support our earlier studies showing attachment of droplets to intermediate filaments, which suggests a role for these filaments in intracellular transport of cholesterol.
Endosomal system of Paramecium: coated pits to early endosomes
Authors: Allen Richard D. , Schroeder Christopher C. , Fok Agnes K. 
Published: April 2021

ABSTRACT
A detailed morphological and tracer study of endocytosis via coated pits in Paramecium multimicronucleatum was undertaken to compare endocytic processes in a free-living protozoon with similar processes in higher organisms. Permanent pits at the cell surface enlarge, become coated and give rise to coated vesicles (188±41 nm in diameter) that enclose fluid-phase markers such as horseradish peroxidase (HRP). Both the pits and vesicles are labeled by the immunogold technique when a monoclonal antibody (mAb) raised against the plasma membrane of this cell is applied to cryosections. The HRP is delivered to an early endosome compartment, which also shares the plasma membrane antigen. The early endosome, as shown in quick-freeze deep-etch replicas of chemically unfixed cells, is a definitive non-reticular compartment composed of many individual flattened cisternal units of 0.2 to 0.7 μm diameter, each potentially bearing one or more ∼80-nm-wide coated évaginations. These coated évaginations on the early endosomes contain HRP but are not labeled by the mAb. The coated évaginations pinch off to form a second group of coated vesicles (90±17 nm in diameter), which can be differentiated from those formed from coated pits by their smaller size, absence of plasma membrane antigen and their location somewhat deeper into the cytoplasm. This study shows a striking similarity between protozoons and mammalian cells in their overall early endosomal machinery and in the ability of early endosomes to sort cargo from plasma membrane components. The vesicles identified in this study form two distinct populations of putative shuttle vesicles, pre-endosomal (large) and early endosome-derived vesicles (small), which facilitate incoming and outgoing traffic from the early endosomes.
Cloning and expression of Drosophila HP1 homologs from a mealybug, Pianococcus citri
Authors: Epstein Helen , James Tharappel C. , Singh Prim B. 
Published: April 2021

ABSTRACT
The mealybug chromosome cycle is one of the most dramatic examples of genomic imprinting known. In embryos that are to become male the entire paternal chromosome set becomes heterochromatic and inactive at the blastoderm stage, while the maternal set remains active and euchromatic. HP1 is a protein from Drosophila melanogaster, which binds preferentially to heterochromatin on polytene chromosomes and is likely to be a modifier of position effect variegation. This paper describes the isolation and sequencing of two cDNA clones encoding HP1 homologs from the mealybug, Pianococcus citri. The protein product of the cDNA clone that was closer to HP1 in sequence was expressed as a fusion protein in Eschericia coli, and polyclonal rat antibodies were raised against it. Immunohistochemistry to mealybug squash preparations showed that this protein was a male-specific nuclear protein, but that it was not specifically associated with the heterochromatic set of chromosomes.
Genetic interactions in the control of septation in Schizosaccharomyces pombe
Authors: Marks John , Fankhauser Christian , Simanis Viesturs 
Published: April 2021

ABSTRACT
We have used genetic and molecular techniques to investigate the interactions among genes required for the initiation and regulation of septum formation in Schizosaccharomyces pombe. Our data suggest that the products of the cdc7, cdc11, cdcl4 and cdcl6 genes interact. These activities may regulate the function of the cdcl5 gene product. A model for the control of septation in fission yeast is presented.
Lymphocyte adhesion to high endothelium is mediated by two β1 integrin receptors for fibronectin, α4β1 and α5β1
Authors: Szekanecz Zoltan , Humphries Martin J. , Ager Ann 
Published: April 2021

ABSTRACT
Using a rat model we have previously proposed a role for fibronectin as an adhesive ligand on high endothelial cells (HEC) for recirculating lymphocytes. Lymphocyte adhesion to high endothelial cells was blocked by CS1 peptide (from the type IH connecting segment of fibronectin) and RGD-containing peptides using two different in vitro assays of lymphocyte-HEC recognition, the frozen section assay and cultured HEC. In order to study the receptors utilised by lymphocytes to bind to HEC we have developed a xenogeneic model in which the adhesion of human lymphocytes to HEC cultured from rat lymph nodes is measured. The basic properties of lymphocyte-HEC interaction were retained using human lymphocytes. CS1 peptide and RGD-containing peptides gave similar profiles of inhibition of lymphocyte adhesion as found previously using rat cells.
FACS analysis showed that the majority of peripheral blood lymphocytes expressed two β1 integrin receptors, α4β1 and α5β1, which are known to recognise distinct adhesion domains in fibronectin. A subpopulation of lymphocytes also expressed α3β1, which, like α5β1, has been reported to be an RGD-dependent adhesion receptor for the central cell binding domain of fibronectin. Antiα4 and anti-β5 subunit monoclonal antibodies maximally inhibited adhesion to HEC by 60% and 65%, respectively. Monoclonal antibodies to the common & subunit gave slightly higher inhibition at 70%. These results suggest that lymphocytes employ one or both of two different β1 integrin fibronectin receptors to bind to HEC. The simultaneous or alternate engagement of two fibronectin receptors on the lymphocyte surface by immobilised fibronectin in the endothelial layer may contribute to the stabilisation of adhesive contacts or to the subsequent transendothelial migration of lymphocytes. In contrast to lymphocytes, peripheral blood neutrophils did not express any members of the β1 integrin family. The selective expression of β1 integrins by lymphocytes and not neutrophils contrasted with the widespread distribution of the other homing-associated adhesion molecules, LECAM-1, CD44 and LFA-1, on these two cell types. It is thus possible that the selective expression of β1 integrins regulates the constitutive migration of lymphocytes but not neutrophils into organised lymphoid tissues.