bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induction of repressible acid phosphatase by unsaturated fatty acid in Saccharomyces cerevisiae
Authors: Doi Syuichi , Watanabe Masayasu , Tanabe Kazuyuki , Nakasako Michiko , Yoshimura Masao 
Published: April 2021

ABSTRACT
We studied the induction of acid phosphatase (APase) by fatty acids in Saccharomyces cerevisiae. S. cerevisiae has two types of APase: constitutive and repressible enzymes. The synthesis of the latter APase is normally derepressed by depletion of inorganic phosphate (P1) in the incubation medium. Of the saturated and unsaturated fatty acids tested, linoleic, linolenic and arachidonic acids induced the synthesis of APase even in the presence of a high concentration of P1, whereas palmitic, stearic and oleic acids did not. De novo protein synthesis but not stimulation of secretion of the enzyme was required for the induction. Genetic analyses using plasmids carrying the genes, PHO5 and PHO3, that code for repressible APase and constitutive APase, respectively, showed that linolenic acid induced the formation of repressible APase. Linolenic acid inhibited the uptake of exogenous 32P1 and simultaneously lowered the intracellular level of P1. These circumstances indicate that linolenic acid-induced derepression of respressible APase is primarily caused by a fall in the intracellular level of P1. However, cells that had been preincubated in the presence of a high concentration of P1 produced APase shortly after the addition of linolenic acid. It is, therefore, suggested that, as well as a normal regulatory mechanism for derepression of repressible APase, a mechanism independent of the external level of P1 participates in the induction of repressible APase by linolenic acid.
Retinoic acid-induced inhibition of lung colonization and changes in the synthesis and properties of glycosaminoglycans of metastatic b16 melanoma cells
Authors: Edward Michael , MacKie Rona M. 
Published: April 2021

ABSTRACT
The effect of all-trans retinoic acid on metastatic B16 melanoma lung colonization and synthesis and properties of glycosaminoglycans was examined. Injection of tumour cells, pretreated with 10−6M-retinoic acid or grown to low density, into the tail vein of syngeneic C57 mice produced significantly fewer pulmonary tumours compared to subconfluent control cells. By cochromatography of glycos-aminoglycans isolated from control ([14C]glucosamine-labelled) and 10−6M-retinoic acid-treated ([3H]glucosamine-labelled) cells on DEAE ion-exchange columns, differences in elution profiles were detected. Chondroitin sulphates isolated from retinoic acid-treated cells eluted at a lower salt concentration than those from control cells, while retinoic acid-treated cells synthesised heparan sulphates of a higher charge density than heparans from control cultures. These changes were apparent in both medium and trypsin-releasable fractions. Retinoic acid-treated cultures were seeded so that they were of a similar density to control cultures when harvested, as cell density was shown to affect glycosaminoglycan synthesis, the glycosaminoglycans from low-density cultures having similar properties to those isolated from retinoic acid-treated cultures. Retinoic acid treatment also reduced the overall synthesis of glycosaminoglycans while having little effect on the composition or distribution between medium, trypsin-releasable and cell-associated fractions.
These observed changes in glycosaminoglycans may, in part, be responsible for retinoic acid-induced inhibition of lung colonization, and reduced adhesion to basement membrane components, which we have previously demonstrated.
Soluble proteins as modulators of the exocytotic reaction of permeabilised rat mast cells
Authors: Koffer A. , Gomperts B. D. 
Published: April 2021

ABSTRACT
This study addresses the question of the role of cytoplasmic proteins in exocytosis from permeabilised rat mast cells. We have used two different methods of cell permeabilisation (ATP4− and streptolysin O) to regulate the size of the plasma membrane lesions, and thus to dictate the rate and extent of efflux of the cytosolic proteins, and compared the secretory response of the two preparations. We report evidence for the existence of two factors present in the cytosol, which affect the exocytotic mechanism in opposing manners. One of these is required for the maintenance of cell responsiveness; it is retained for more than 120 min by ATP4−-permeabilised cells but lost within 60 min from cells permeabilised by streptolysin O. The other factor, which leaks immediately from cells treated from streptolysin O, but only gradually from cells treated with ATP4−, has the effect of suppressing the affinity for both Ca2+ and guanine nucleotide in the exocytotic reaction.
Sequestrocytes: a manifestation of transcellular cross-bonding of the red cell membrane in sickle cell anemia
Authors: Weinstein Ronald S. , Warth James A. , Near Karen , Marikovsky Yehuda 
Published: April 2021

ABSTRACT
An unusual form of red cells (called sequestrocytes) that circulate in the peripheral blood of patients with sickle cell anemia has been identified. In wet-mount light microscopy preparations, sequestro-cytes appear as massively vacuolated erythrocytes. We have shown by transmission electron microscopy and scanning electron microscopy that the morphology of sequestrocytes can be accounted for on the basis of transcellular cross-bonding of the cell membrane. Initially, endocytotic vesicles span the cytoplasmic space, forming putative cytoskeletal fusion zones. Points of fusion of the membrane skeleton bridging the cytoplasmic compartment expand laterally to form linear fusion zones that entrap lakes of hemoglobin within pseudo vacuoles.
Sequestrocytes can be isolated in the densest layer (1.159 g ml−1 or greater) of an arabinogalactan density gradient. These cells can be generated in increased numbers in sickle cell blood by incubating samples in 1.5 mM-acetylphenylhydrazine (APH) solution for two hours at 37°C. Their formation is partially blocked by incubation with the reducing agent, dithiothreitol (DTT). Our results suggest that these cells represent an expression of oxidative membrane injury in sickle cell anemia.
Confocal fluorescence microscopy with the tandem scanning light microscope
Authors: Wright Shirley J. , Walker James S. , Schatten Heide , Simerly Calvin , Mccarthy Jon J. , Schatten Gerald 
Published: April 2021

ABSTRACT
Applications of the tandem scanning confocal microscope (TSM) to fluorescence microscopy and its ability to resolve fluorescent biological structures are described. The TSM, in conjunction with a cooled charge-coupled device (cooled CCD) and conventional epifluorescence light source and filter sets, provided high-resolution, confocal data, so that different fluorescent cellular components were distinguished in three dimensions within the same cell. One of the unique features of the TSM is the ability to image fluorochromes excited by ultraviolet light (e.g. Hoechst, DAPI) in addition to fluorescein and rhodamine. Since the illuminationis dim, photobleaching is insignificant and prolonged viewing of living specimens is possible. Series of optical sections taken in the Z-axis with the TSM were reproduced as stereo images and threedimensional reconstructions. These data show that the TSM is potentially a powerful tool in fluorescence microscopy for determining three-dimensional relationships of complex structures within cells labeled with multiple fluorochromes.
Actin is associated with the formation of the cell wall in reverting protoplasts of the fission yeast Schizosaccharomyces pombe
Authors: Kobori Hiromi , Yamada Naoko , Taki Akiko , Osumi Masako 
Published: April 2021

ABSTRACT
To clarify the involvement of actin in the formation of the yeast cell wall, reverting protoplasts of Schizosaccharomyces pombe were used as a simple model system. Actin of reverting protoplasts was labeled with rhodamine-conjugated phalloidin and observed by conventional fluorescence microscopy and laser scanning confocal microscopy. A close spatial as well as temporal relationship between actin and cell wall formation was observed in protoplast reversion. That is, the site of actin‘dots’ in the reverting protoplasts coincided with the site of new wall formation and the timing of rearrangement of actin coincided with the initiation of cell wall formation and with the timing of cell wall expansion.
Treatment of reverting protoplasts with cytochalasin D (CD) further clarified the close relationship between actin and cell wall organization. The effect of CD was dose dependent. A high dose of CD caused the absence of actin as well as the complete inhibition of cell wall formation. A low dose of CD caused weakly stained unlocalized actin, which induced grossly aberrant cell wall deposition as well as substantial changes in the morphology of the reverting protoplasts. These results demonstrated that actin is associated with initiation of cell wall formation, the proper deposition of cell wall materials, and maintaining the normal morphology of reverting protoplasts. Scanning electron microscopy revealed the presence of a fibrillar net structure on the surface of non-treated control reverting protoplasts. However, the absence of a fibrillar network on the surface of reverting protoplasts was observed in the presence of a high concentration of CD. Lack of localization of microfibrils as well as poor development of the fibrillar network were also observed in the presence of a low concentration of CD. Recovery experiments confirmed the close relationship between actin and cell wall formation.
Microtubule dynamic instability: Numerical simulation of microtubule transition properties using a lateral cap model
Authors: Bayley Peter M. , Schilstra Maria J. , Martin Stephen R. 
Published: April 2021

ABSTRACT
We present a numerical formulation for the dynamic instability of microtubules involving the stabilisation of growing microtubules by a single layer of tubulin-GTP, with GTP hydrolysis effectively coupled to tubulin-GTP addition. This Lateral Cap model provides a readily visualised, working mechanism for the co-existence and interconversion of growing and shrinking microtubules.
This class of model is specified in terms of a hydrolysis rule, whereby the addition of tubulin-GTP causes hydrolysis of GTP on a previously terminal tubulin-GTP molecule as it becomes incorporated into the microtubule lattice. A specific formulation is illustrated, though this is not unique. A limited set of parameters defines the kinetics and affinity for tubulin-GTP at the binding sites at a given end of the microtubule. The rate constants are a function of the nucleotide composition of the binding site, principally comprising the two tubulin molecules, which interact laterally and longitudinally with the incoming tubulin-GTP molecule.
The Lateral Cap formulation demonstrates that a single terminal layer of tubulin-GTP is sufficient to reproduce the apparently complex behaviour of a dynamic population of microtubules. It differs significantly from the fluctuating tubulin-GTP cap model of Chen and Hill (1985). It gives a molecular description to the switching of individual microtubules between growing and shrinking states in terms of the composition of the multi-start terminal layer of the microtubule, and provides a general mechanism for the differential kinetic behaviour at opposite ends of dynamic microtubules. It reproduces the essential features of microtubule length excursions, and predicts detailed characteristics of microtubule dynamics, including the basis of the apparently cooperative nature of the transition behaviour as a function of the concentration of tubulin-GTP. It is readily amenable to further experimental test and refinement.
Laminin interactions with ductal pancreatic adenocarcinoma cells: Identification of laminin- and collagen-binding proteins
Authors: Mai M. , Brune K. , Jacoby B. , Kern H. F. , Mollenhauer J. 
Published: April 2021

ABSTRACT
Laminin promotes the modulation of human pancreatic adenocarcinoma cells from a proliferative to a resting phenotype. This process includes restoration of cell polarity, increase of protein biosynthesis, and increase of glycoprotein secretion. The growth correlates with the amount of laminin coated on the culture dish. Adenocarcinoma cells do not synthesize collagen type I, fibronectin or laminin. Prolonged propagation of the cells on laminin substratum enhances the expression of laminin-binding sites on the cell surface. Laminin binds to cell plasma membrane vesicles with a KB of about 2.2×1010. By affinity chromatography of [35S]methionine-labeled, detergent-extracted, cells on immobilized laminin, a Mr82000 polypeptide could be enriched. By simultaneous chromatography on immobilized collagen type I, a Mr34000 polypeptide was retained. In laminin overlay experiments on transblotted plasma membrane proteins, Mr82000 and 70000 polypeptides were labeled. Affinity chromatography on laminin-Sepharose of tumor cell membranes from cells grown in nude mice tumors retained Mr 100 000, 82000, 70000 and 55000 polypeptides bound to the column. Endoglycosidase F treatment of these proteins reduced the number of higher Mr proteins, leaving a Mr 70 000 polypeptide, together with smaller peptides. Using the enriched binding protein fraction as antigens, monoclonal antibodies (mAbs) were prepared in mice. Two of the mAbs were further analysed; they recognized simultaneously the complete pattern of high and low Mr polypeptides identified to date by the other methods. Antibody 2A1-H7 was capable of inhibiting attachment and spreading of the cells on laminin and collagen I, but not on tissue-culture plastic. These data may indicate a molecular heterogeneity, partly based on diverse glycosylation, responsible for the variations in the molecular weight of the binding proteins. The adaptation processes of the tumor cells during growth on the extracellular matrix may indicate a regulatory function on tumor growth and metastasis in vivo.
Monoclonal antibody against a conjugation-specific nuclear antigen in Paramecium caudatum
Authors: Yanagi Akira , Yamamoto Hiroaki 
Published: April 2021

ABSTRACT
To understand molecular mechanisms controlling the sexual reproduction process (conjugation) in Paramecium caudatum, we have tried to detect conjugation-specific antigens with monoclonal antibodies. We obtained a monoclonal antibody (CSN-1) against an antigen that appears in the nuclei only during conjugation. This nuclear antIgen began to appear both in microand macronuclei at micronuclear stage II or HI early in the conjugation process (4 h after the mating reaction at 25°C). In the macronucleus, the nuclear antigen persisted until the stage of macronuclear fragmentation (about 35 h) and then disappeared before degeneration of the macronuclear fragments. In the micronucleus, the antigen existed until the crescent stage (stage V) of the first meiotic division (8h). The antigen in the micronucleus disappeared after the crescent stage but reappeared again in the eight nuclei produced by the third postzygotic division (25 h). Then it persisted only in the four macronuclear anlagen differentiated from the eight nuclei (about 30 h). When exconjugant cells had undergone two successive postconjugational cell divisions and thus possessed only one new macronucleus as in the vegetative cells, the antigen disappeared completely from the new macronucleus in most cells. These cells without the antigen began to appear about 50 h after the mating reaction. As the antigen is specific to conjugation and localized in nuclei, it may play some important role(s) in the conjugation process.
Poly-N-acetyllactosamine-specific tomato lectin interacts with gastric parietal cells Identification of a tomato-lectin binding 60-90×103/Mr membrane glycoprotein of tubuloveslcles
Authors: Callaghan Judy M. , Toh Ban-Hock , Pettitt Ohn M. , Humphris Danielle C. , Gleeson Paul A. 
Published: April 2021

ABSTRACT
The cytoplasmic tubulovesicular and canalicular membranes of gastric parietal cells are intimately involved in hydrochloric acid secretion. To characterise the glycoproteins of these membranes, we examined a panel of lectins for reactivity with parietal cells in paraffin sections of rat, dog and pig stomach. The poly-N-acetyllactosamine-specific lectin from Lycopersicon esculentum (tomato) and from Solatium tuberosum (potato), and the galactose-specific lectin Ricinus communis agglutinin (RCAt2o), showed strong cytoplasmic binding of parietal cells of all three species, with a pattern indicative of an intracellular membrane network. Binding to parietal cells was confirmed by doublelabelling studies with parietal cell auto-antibodies from patients with autoimmune gastritis. Mucous cells and mucin also bound these lectins strongly. Other gastric cell types did not stain with either tomato or potato lectin, but stained weakly with RCA12O-Electron-microscopic examination of lectin binding sites using biotinylated tomato lectin or RCA120 and streptavidin-gold, revealed specific binding to the luminal face of parietal cell tubulovesicular and canalicular membranes as well as the contents of mucous cell secretory granules.
Tomato lectin and RCA120 reacted by lectin blotting with a major species of apparent molecular weight 60-90×103Mr from rat, dog and pig gastric membranes. A tubulovesicular membrane fraction, enriched 10-fold for K+-dependent phosphatase activity, was also enriched three-fold for tomato lectin binding as assessed by a solid-phase lectin assay. The 60-90K (K=103Mr) component, in 125I-labelled detergent extracts of dog tubulovesicular membranes, bound to an affinity support of tomato lectin-Sepharose and was specifically eluted with N,AT,AT’-triacetylchitotriose. Digestion with N-glycanase collapsed the 60-90K component into a sharp 35K band.
We conclude that: (1) a 60-90K membrane glycoprotein localised on the luminal face of tubulovesi-cles and canaliculi of parietal cells interacts strongly with tomato lectin and RCA120; and (2) the glycoprotein is composed of a 35K core protein glycosylated with N-glycans probably containing poly-N-acetyllactosamine sequences with terminal galactosyl residues. The properties of this 60-90K glycoprotein are identical to a major parietal cell autoantigen recognised by sera of patients with autoimmune gastritis. As this molecule represents the only component other than the 95K catalytic subunit of the H+/K+-ATPase, which has been specifically identified with parietal cell tubulovesi-cles, and as it has a similar sized protein core to that of the β subunit of the Na+/K+-ATPase, we propose that it is the β subunit of the proton pump.
Keratinocyte transglutaminase in human skin and oral mucosa: cytoplasmic localization and uncoupling of differentiation markers
Authors: Ta Bach Mai , Gallagher George T. , Chakravarty Rupa , Rice Robert H. 
Published: April 2021

ABSTRACT
Expression of kératinocyte transglutaminase, a specific differentiation marker, has been examined by immunogold-silver cytochemistry in human epidermis and oral epithelium, and in oral mucosal hyperplasia and neoplasia. Two major findings have been obtained. First, considerable immunoreactivity was evident not only at the plasma membrane (the site of cross-linked envelope formation) but also in the cytoplasm of spinous cells, suggesting a cytoplasmic function for this transglutaminase. Staining at the cell border was seen principally in the granular layer of orthokeratinized epithelium (epidermis, hard palate), the outer spinous cells of ortho- and parakeratinized epithelium and in the suprabasal cells showing squamous differentiation in benign and malignant neoplasms. By contrast, diffuse cytoplasmic staining was observed in the upper spinous layer of the normal epithelium and benign lesions. The cytoplasmic immunoreactivity, which extended nearly to the basal layer in hyperkeratosis of the oral mucosa, was evident in two of three verrucous carcinomas examined. In keeping with their undifferentiated character, invasive nests of squamous cell carcinoma and basaloid epithelium in benign and neoplastic lesions were immunonegative for transglutaminase. The second major finding was that lesions of severe oral epithelial dysplasia, immunonegative for transglutaminase, were capable of expressing involucrin immunoreactivity, indicating an uncoupling of keratinocyte programming. These results suggest that immunogold-silver staining for transglutaminase may be useful in evaluating the degree of differentiation in benign and malignant oral epithelial proliferation.
Role of nuclear protein kinase C in the mitogenic response to platelet-derived growth factor
Authors: Fields Alan P. , Tyler Greg , Kraft Andrew S. , May W. Stratford 
Published: April 2021

ABSTRACT
We have assessed the involvement of nuclear envelope protein phosphorylation in the mitogenic response to platelet-derived growth factor (PDGF) in NIH/3T3 fibroblasts. We find that stimulation of quiescent NIH/3T3 cells with PDGF or with the mitogenic protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA) or bryostatin 1 (bryo) leads to rapid, dose-dependent phosphorylation of several nuclear envelope polypeptides. The predominant nuclear envelope targets for mitogen-induced phosphorylation are immunologically identified as the nuclear envelope lamins. All three lamin species (A, B and C) are phosphorylated in response to PMA or bryo, while lamins A and C are preferentially phosphorylated in response to PDGF. Phosphopeptide mapping and phosphoamino acid analysis indicate that similar serine sites on the lamins are phos phorylated in response to PDGF, PMA and bryo. Both mitogenicity and lamina phosphorylation induced by these mitogens can be inhibited by the selective PKC inhibitor staurosporine at 2 nM. Treatment of quiescent NLH/3T3 cells with PDGF, PMA or bryo leads to rapid translocation of PKC to the nuclear envelope. These data indicate that rapid nuclear events, including translocation of cytosolic PKC to the nuclear membrane and lamina phosphorylation, may play a role in the transduction of the mitogenic signals of PDGF from the cytoplasm to the nucleus in NTH/3T3 fibroblasts.
Binding of human endothelium to Ulex europaeus l-coated Dynabeads: application to the isolation of microvascular endothelium
Authors: Jackson C. J. , Garbett P. K. , Nissen B. , Schrieber L. 
Published: April 2021

ABSTRACT
A major problem encountered when isolating human microvascular endothelium is the presence of contaminating cells such as fibroblasts that rapidly over-grow the endothelial cells. We describe here a simple, rapid technique for purifying endothelial cells derived from the microvasculature of neonatal foreskin and osteoarthritic and rheumatoid arthritic synovium. This technique is based on the selective binding of the lectin Ulex europaeus I (UEA I) to the endothelial cell surface via fucose residues. Initially UEA I was covalently bound to tosyl-activated super-paramagnetic polystyrene beads (Dynabeads) by incubation for 24 h at room temperature. Cells were isolated by extracting microvascular segments from enzyme-treated (trypsin and Pronase) cubes of tissue. The mixed population of cells obtained were purified by incubating them at 4 °C for 10 min with the UEA I-coated Dynabeads. Endothelium bound to the beads whilst contaminating cells were removed by five washes with HBSS using a magnetic particle concentrator. The endothelial cells thus obtained grew to confluence as a cobblestone-like monolayer and expressed von Willebrand factor antigen. The cells were released from the Dynabeads by the competitive binding of fucose (10 min at 4°C). This new method is simple and reproducible and allows pure human microvascular endothelial cells to be cultured within 2 h of obtaining a specimen.