scholarly journals Release of hyaluronate from eukaryotic cells

1990 ◽  
Vol 267 (1) ◽  
pp. 185-189 ◽  
Author(s):  
P Prehm
Keyword(s):  
Cell Lines ◽  
Glucuronic Acid ◽  
Gel Filtration ◽  
Eukaryotic Cell ◽  
Chain Elongation ◽  
Eukaryotic Cells ◽  
Radical Scavengers ◽  
Mass Distributions ◽  
Partial Degradation ◽  
Extension Period

The mechanism of hyaluronate shedding from eukaryotic cell lines was analysed. All cell lines shed identical sizes of hyaluronate as were retained on the surface. They differed in the amount of hyaluronate synthesized and in the proportions of hyaluronate which were released and retained. A method was developed which could discriminate between shedding due to intramolecular degradation and that due to dissociation as intact macromolecules. This method was applied to B6 and SV3T3 cells in order to study the mechanism of hyaluronate release in more detail. The cells were pulse-labelled to form hyaluronate chains with labelled and unlabelled segments, and the sizes of labelled hyaluronate released into the medium during the pulse extension period were determined by gel filtration. B6 cells released identical sizes of hyaluronate at all labelled segment lengths, indicating that no intramolecular degradation occurred. When chain elongation was blocked by periodate-oxidized UDP-glucuronic acid, hyaluronate release was simultaneously inhibited. These results indicated that B6 cells dissociated hyaluronate as an intact macromolecule. In contrast, SV3T3 cells released hyaluronate of varying molecular mass distributions during extension of the labelled segment, suggesting partial degradation. Exogenous hyaluronate added to SV3T3 cultures was also degraded. This degradation could be prevented by the presence of radical scavengers such as superoxide dismutase and tocopherol. Degradation of endogenous hyaluronate could be inhibited by salicylate. These results led to the conclusion that SV3T3 cells released hyaluronate not only by dissociation, but also by radical-induced degradation.

Assay for Adherence of Vibrio cholerae to Eukaryotic Cell Lines

BIO-PROTOCOL ◽  
2014 ◽  
Vol 4 (8) ◽  
Author(s):  
Amit Dey ◽  
Abha Bhagat ◽  
Rukhsana Chowdhury
Keyword(s):  
Vibrio Cholerae ◽  
Cell Lines ◽  
Eukaryotic Cell

scholarly journals Origin of the Eukaryotic Cell

2017 ◽  
Vol 01 (02) ◽  
pp. 108-120 ◽  
Author(s):  
Nick Lane
Keyword(s):  
Protein Synthesis ◽  
Host Cell ◽  
Genome Size ◽  
Energy Savings ◽  
Nuclear Genome ◽  
Eukaryotic Cell ◽  
Eukaryotic Cells ◽  
Bacterial Genomes ◽  
Complex Cells ◽  
Life On Earth

All complex life on Earth is composed of ‘eukaryotic’ cells. Eukaryotes arose just once in 4 billion years, via an endosymbiosis — bacteria entered a simple host cell, evolving into mitochondria, the ‘powerhouses’ of complex cells. Mitochondria lost most of their genes, retaining only those needed for respiration, giving eukaryotes ‘multi-bacterial’ power without the costs of maintaining thousands of complete bacterial genomes. These energy savings supported a substantial expansion in nuclear genome size, and far more protein synthesis from each gene.

Eukaryotic cell lines as a sensitive layer for direct monitoring of carbon monoxide

2011 ◽  
Vol 208 (6) ◽  
pp. 1345-1350 ◽  
Author(s):  
Ulrich Bohrn ◽  
Evamaria Stütz ◽  
Maximilian Fleischer ◽  
Michael J. Schöning ◽  
Patrick Wagner
Keyword(s):  
Carbon Monoxide ◽  
Cell Lines ◽  
Eukaryotic Cell ◽  
Sensitive Layer

Immunofluorescent localization of the ubiquitin-activating enzyme, E1, to the nucleus and cytoskeleton

1993 ◽  
Vol 264 (1) ◽  
pp. C93-C102 ◽  
Author(s):  
J. S. Trausch ◽  
S. J. Grenfell ◽  
P. M. Handley-Gearhart ◽  
A. Ciechanover ◽  
A. L. Schwartz
Keyword(s):  
Cell Lines ◽  
Chinese Hamster ◽  
Eukaryotic Cell ◽  
Nuclear Staining ◽  
Immunofluorescent Localization ◽  
Acid Protein ◽  
Ubiquitin Conjugation ◽  
Double Label ◽  
Pleiotropic Functions ◽  
Amino Acid Protein

Ubiquitin, a 76-amino acid protein, is covalently attached to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Ubiquitin is found within the cytoplasm, nucleus, microvilli, autophagic vacuoles, and lysosomes. The ubiquitin-activating enzyme, E1, catalyzes the first step in ubiquitin conjugation. To date, very little is known about the subcellular distribution of this enzyme. We have utilized immunofluorescence and immunoblotting to examine the cellular distribution of E1 in several eukaryotic cell lines, including HeLa, smooth muscle A7r5, choriocarcinoma BeWo, Pt K1, and Chinese hamster ovary (CHO) E36. E1 was identified in both cytoplasmic and nuclear compartments in all cell lines examined. However, the relative abundance within these compartments differed markedly between the cell lines. Even within a single cell line, nuclear distribution was not uniform, and certain cells demonstrated an absence of nuclear staining. E1 resides predominantly within the nucleus in BeWo. In contrast, its distribution in CHO and Pt K1 cells is mainly cytoplasmic. Within the cytoplasm, three pools of E1 were identified by double-label immunofluorescence. The first of these colocalized with phalloidin, indicating association of E1 with actin filaments. A second cytoplasmic pool colocalized with tubulin and was predominantly perinuclear in its distribution. The third pool associated with intermediate filaments. This suggests that E1 is associated with all three components of the cytoskeleton. The distribution of E1 was unaltered in a mutant line of CHO E36 designated ts20, in which the E1 can be thermally inactivated. The variable distribution of E1 among cell lines, including its apparent cytoskeletal association, suggests pleiotropic functions of this enzyme and the ubiquitin-conjugating system.

scholarly journals Bacterial tail anchors can target to the mitochondrial outer membrane

2017 ◽  
Author(s):  
Güleycan Lutfullahoğlu Bal ◽  
Abdurrahman Keskin ◽  
Ayşe Bengisu Seferoğlu ◽  
Cory D. Dunn
Keyword(s):  
Outer Membrane ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Eukaryotic Cell ◽  
Eukaryotic Cells ◽  
Mitochondrial Outer Membrane ◽  
Bacterial Proteins ◽  
Rate Limiting Step ◽  
Protein Entry ◽  
Rate Limiting

ABSTRACTDuring the generation and evolution of the eukaryotic cell, a proteobacterial endosymbiont was refashioned into the mitochondrion, an organelle that appears to have been present in the ancestor of all present-day eukaryotes. Mitochondria harbor proteomes derived from coding information located both inside and outside the organelle, and the rate-limiting step toward the formation of eukaryotic cells may have been development of an import apparatus allowing protein entry to mitochondria. Currently, a widely conserved translocon allows proteins to pass from the cytosol into mitochondria, but how proteins encoded outside of mitochondria were first directed to these organelles at the dawn of eukaryogenesis is not clear. Because several proteins targeted by a carboxyl-terminal tail anchor (TA) appear to have the ability to insert spontaneously into the mitochondrial outer membrane (OM), it is possible that self-inserting, tail-anchored polypeptides obtained from bacteria might have formed the first gate allowing proteins to access mitochondria from the cytosol. Here, we tested whether bacterial TAs are capable of targeting to mitochondria. In a survey of proteins encoded by the proteobacterium Escherichia coli, predicted TA sequences were directed to specific subcellular locations within the yeast Saccharomyces cerevisiae. Importantly, TAs obtained from DUF883 family members ElaB and YqjD were abundantly localized to and inserted at the mitochondrial OM. Our results support the notion that eukaryotic cells are able to utilize membrane-targeting signals present in bacterial proteins obtained by lateral gene transfer, and our findings make plausible a model in which mitochondrial protein translocation was first driven by tail-anchored proteins.

scholarly journals Interleukin-HP1, a T cell-derived hybridoma growth factor that supports the in vitro growth of murine plasmacytomas.

1987 ◽  
Vol 165 (3) ◽  
pp. 641-649 ◽  
Author(s):  
J Van Snick ◽  
A Vink ◽  
S Cayphas ◽  
C Uyttenhove
Keyword(s):  
T Cell ◽  
Growth Factor ◽  
B Cell ◽  
Cell Lines ◽  
Primary Cultures ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Hybridoma Growth ◽  
Cell Supernatant

We have recently described the purification and NH2-terminal amino acid sequence of a T cell-derived hybridoma growth factor that was provisionally designated interleukin-HP1 (IL-HP1). Here we report that a T cell supernatant containing high titers of this hybridoma growth factor considerably facilitated the establishment of primary cultures of murine plasmacytomas. Most plasmacytoma cell lines derived from such cultures remained permanently dependent on IL-HP1-containing T cell supernatant for both survival and growth in vitro. These cell lines, however, retained their ability to form tumors in irradiated pristane-treated mice. Analytical fractionation of a T cell supernatant rich in IL-HP1 by either gel filtration, isoelectric focusing, or reversed-phase HPLC revealed the existence of only one plasmacytoma growth factor activity that strictly copurified with IL-HP1, strongly suggesting the identity of both factors. This conclusion was further supported by the finding that IL-HP1 purified to homogeneity supported the growth of both B cell hybridomas and plasmacytomas. For half-maximal growth, plasmacytomas, however, required a concentration of IL-HP1 of approximately 30 pM, which is approximately 200 times higher than that required by B cell hybridomas. A clear difference in the specificity of IL-HP1 and B cell stimulatory factor 1 (BSF-1) was demonstrated by the finding that IL-HP1-dependent plasmacytomas did not survive in the presence of BSF-1, whereas helper T cell lines that proliferated in the presence of BSF-1 failed to respond to IL-HP1.

scholarly journals Listeria monocytogenes Possesses Adhesins for Fibronectin

1999 ◽  
Vol 67 (12) ◽  
pp. 6698-6701 ◽  
Author(s):  
Philippe Gilot ◽  
Paul André ◽  
Jean Content
Keyword(s):  
Extracellular Matrix ◽  
Listeria Monocytogenes ◽  
Cell Surface ◽  
Bacterial Cell ◽  
Cell Types ◽  
Eukaryotic Cell ◽  
Eukaryotic Cells ◽  
Food Borne ◽  
Human Fibronectin ◽  
Fibronectin Binding

ABSTRACT Listeria monocytogenes is a gram-positive, nonsporulating, food-borne pathogen of humans and animals that is able to invade many eukaryotic cells. Several listerial surface components have been reported to interact with eukaryotic cell receptors, but the complete mechanism by which the bacteria interact with all of these cell types remains largely unknown. In this work, we found thatL. monocytogenes binds to human fibronectin, a 450,000-Da dimeric glycoprotein found in body fluids, on the surface of cells and in an insoluble component of the extracellular matrix. The binding of fibronectin to L. monocytogenes was found to be saturable and dependent on proteinaceous receptors. Five fibronectin-binding proteins of 55.3, 48.6, 46.7, 42.4, and 26.8 kDa were identified. The 55.3-kDa protein was proved to be present at the bacterial cell surface. The binding of L. monocytogenes to fibronectin adds to the number of molecules to which the bacterium is able to adhere and emphasizes the complexity of host-pathogen interactions.

scholarly journals The binding of oxidized and reduced nicotinamide–adenine dinucleotides to bovine liver uridine diphosphate glucose dehydrogenase

1974 ◽  
Vol 141 (3) ◽  
pp. 667-673 ◽  
Author(s):  
Paul A. Gainey ◽  
Charles F. Phelps
Keyword(s):  
Glucuronic Acid ◽  
Glucose Dehydrogenase ◽  
Gel Filtration ◽  
Bovine Liver ◽  
Uridine Diphosphate ◽  
Protein Fluorescence ◽  
Diphosphate Glucose ◽  
Ph Dependent ◽  
Uridine Diphosphate Glucose Dehydrogenase ◽  
Biphasic Profile

The binding of NAD+and NADH to bovine liver UDP-glucose dehydrogenase was studied by using gel-filtration and fluorescence-titration methods. The enzyme bound 0.5mol of NAD+and 2 mol of NADH/mol of subunit at saturating concentrations of both substrate and product. The dissociation constant for NADH was 4.3μm. The binding of NAD+to the enzyme resulted in a small quench of protein fluorescence whereas the binding of NADH resulted in a much larger (60–70%) quench of protein fluorescence. The binding of NADH to the enzyme was pH-dependent. At pH8.1 a biphasic profile was obtained on titrating the enzyme with NADH, whereas at pH8.8 the titration profile was hyperbolic. UDP-xylose, and to a lesser extent UDP-glucuronic acid, lowered the apparent affinity of the enzyme for NADH.

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