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At least 19 recordsLinked to original sources

How transcription factors regulate origins of DNA replication in eukaryotic cells.

Eukaryotic chromosomes contain a few thousand origins of DNA replication, which are activated in a temporal and spatial order during S phase. One parameter that is strongly implicated in determining the order of replication is transcription. This review focuses on the role of transcription factors in activating origins of replication in eukaryotic cells. Studies of viral and mitochondrial replication origins have revealed several mechanisms by which transcription factors activate origins, but it remains to be seen whether any of these are used to regulate cellular chromosome replication.

Journal Article↗

The origin of eukaryotes: the difference between prokaryotic and eukaryotic cells.

Eukaryotes have long been thought to have arisen by evolving a nucleus, endomembrane, and cytoskeleton. In contrast, it was recently proposed that the first complex cells, which were actually proto-eukaryotes, arose simultaneously with the acquisition of mitochondria. This so-called symbiotic association hypothesis states that eukaryotes emerged when some ancient anaerobic archaebacteria (hosts) engulfed respiring alpha-proteobacteria (symbionts), which evolved into the first energy-producing organelles. Therefore, the intracellular compartmentalization of the energy-converting metabolism that was bound originally to the plasma membrane appears to be the key innovation towards eukaryotic genome and cellular organization. The novel energy metabolism made it possible for the nucleotide synthetic apparatus of cells to be no longer limited by subsaturation with substrates and catalytic components. As a consequence, a considerable increase has occurred in the size and complexity of eukaryotic genomes, providing the genetic basis for most of the further evolutionary changes in cellular complexity. On the other hand, the active uptake of exogenous DNA, which is general in bacteria, was no longer essential in the genome organization of eukaryotes. The mitochondrion-driven scenario for the first eukaryotes explains the chimera-like composition of eukaryotic genomes as well as the metabolic and cellular organization of eukaryotes.

Animals↗

[The origin of the eukaryotic cell. III. Principles of the morphofunctional organization of the eukaryotic cell].

The eukaryotic plasmalemma, eukaryotic cytoplasm with its usual cytomembranes, and eukaryotic nucleus are obligatory components of the eukaryotic cell. All other structural elements (organelles) are only derivates of the aforesaid cell components and they may be absent sometimes. There are protozoans having simultaneously no flagelles, mitochondria and chloroplasts (all the representatives of phylum Microspora, amoeba Pelomyxa palustris, and others). The following five general principles play the main role in the morphofunctional organization of the cell. The principle of hierarchy of block organization of living systems. Complex morphofunctional blocks (organelles) specific for the eukaryotic cell are formed. The compartmentalization principle. The main cell organelles (nuclei, flagellae, mitochondria, chloroplasts, etc.) undergo a relative morphological isolation from each other and other cell organelles by means of the total or partial surrounding by membranes; this may ensure the originality of their evolution and function. The principle of poly- and oligomerization of morphofunctional blocks. It permits the cell to enlarge its sizes and to raise the level of integration. The principle of heterochrony, including three subprinciples: conservatism of useful signs; a strong acceleration of evolutionary development of the separate blocks; simplification of the structure, reduction or total disappearance of some blocks. It explains a preservation of prokaryotic signs in the eukaryotic cell or in its organelles. The principle of independent origin of similar morphofunctional blocks in the process of evolution of living systems. The parallelism of the signs in unrelated groups of cells (or protists) arises due to this principle.

Animals↗

Identification of Salmonella functions critical for bacterial cell division within eukaryotic cells.

Salmonella typhimurium multiplication inside eukaryotic host cells is critical for virulence. Salmonella typhimurium strain SL1344 appears as filaments upon growth in macrophages and MelJuSo cells, a human melanoma cell line, indicating a specific blockage in the bacterial cell division process. Several studies have investigated the host cell response impairing bacterial division. However, none looked at the bacterial factors involved in inhibition of Salmonella division inside eukaryotic cells. We show here that blockage in the bacterial division process is sulA-independent and takes place after FtsZ-ring assembly. Salmonella typhimurium genes in which mutations lead to filamentous growth within host cells were identified by a large scale mutagenesis approach on strain 12023, revealing bacterial functions crucial for cell division within eukaryotic cells. We finally demonstrate that SL1344 filamentation is a result of hisG mutation, requires the activity of an enzyme of the histidine biosynthetic pathway HisFH and is specific for the vacuolar environment.

Aminohydrolases↗

An investigation of the ability of TSH and Graves' immunoglobulin G to increase intracellular calcium in human thyroid cells, rat FRTL-5 thyroid cells and eukaryotic cells transfected with the human TSH receptor.

The purpose of this study was to determine if immunoglobulin G preparations (IgGs) from patients with Graves' disease can increase intracellular calcium in thyroid cells, as has been reported for TSH. Both TSH and Graves' IgGs (prepared by protein G affinity chromatography) increased calcium in a range of thyroid cells; however, the response seen, using Fura-2-loaded coverslips of cell monolayers, varied considerably. Chinese hamster ovary (CHO/JPO9) cells transfected with a high number of human TSH receptors showed the greatest response: TSH (10 mU/ml) increased calcium in 46% of experiments and 18 out of 25 (72%) Graves' IgGs increased calcium at 0.1 mg/ml (significantly greater, P < 0.001, than for control IgGs where cells responded to 2 out of 13 preparations). Rat FRTL-5 cells only responded to TSH in 22% of experiments and to 2 out of 8 (25%) of Graves' IgGs. Similarly, human thyroid cells responded to TSH in 22% of experiments and to 2 out of 9 (22%) of Graves' IgGs. (When studying cyclic AMP responses in JPO9 cells, much higher concentrations of Graves' IgGs were required (1-3 mg/ml). However, higher concentrations (0.3 mg/ml) of both Graves' IgGs, and to a lesser extent of control IgGs, were capable of increasing calcium in cells both with and without TSH receptors (control CHO cells and normal human dermal fibroblasts). We conclude that relatively low concentrations of patient IgGs can be distinguished from control IgGs in JPO9 cells on the basis of their ability to increase calcium, but that additionally all IgG preparations possibly contain another factor which can increase calcium in a range of cells independent of the presence of the TSH receptor.

Adolescent↗

Intracellular expression of the Salmonella plasmid virulence protein, SpvB, causes apoptotic cell death in eukaryotic cells.

The spv genes carried on the Salmonella virulence plasmid are commonly associated with severe systemic infection in experimental animals. The SpvB virulence-associated protein has been shown to ADP-ribosylate actin, and this enzymatic activity is essential for virulence in mice. Here, we present evidence that intracellular expression of SpvB protein induces not only disruption of actin filaments but also apoptotic cell death in eukaryotic cells.

3T3 Cells↗

Vesicular transport: how many Ypt/Rab-GTPases make a eukaryotic cell?

In eukaryotic cells, protein transport through the secretory and endocytic pathways is mediated by vesicular intermediates. Individual transport steps are regulated by Ras-like guanine nucleotide-binding proteins, termed Ypt in yeast or Rab in mammals. The complete sequencing of the Saccharomyces cerevisiae genome has revealed the total number of Ypt GTPases in this organism. There is some redundancy among the 11 Ypt proteins, and only those involved in the biosynthetic pathway are essential for cell viability.

Amino Acid Sequence↗

Spermidine requirement for cell proliferation in eukaryotic cells: structural specificity and quantitation.

Six structural homologs of spermidine and five of its precursor, putrescine, were studied for their ability to prevent cytostasis of cultured L1210 leukemia cells induced by alpha-difluoromethylornithine (DFMO), a specific inhibitor of putrescine biosynthesis. High-performance liquid chromatography and competition studies with spermidine indicated that the homologs, which vary in the length of the carbon chain separating the amines, penetrated the cells. The structural specificity of the spermidine carrier was defined. Three of the six spermidine homologs supported cell growth during a 48-hour incubation in the presence of DFMO, indicating that a two-carbon extension of spermidine structure was tolerated for biological function. Two of the five putrescine homologs supported growth after being converted by the cells to their respective spermidine homologs. The central nitrogen of spermidine appears to be essential for function since diamines of chain length comparable to that of spermidine did not prevent DFMO cytostasis. No more than 15 percent of the spermidine normally present in L1210 cells was required for cell proliferation in the presence of DFMO.

Animals↗

The Schaechter-Bentzon-Maaløe experiment and the analysis of cell cycle events in eukaryotic cells.

The Schaechter-Bentzon-Maaløe (SBM) experiment, performed more than 40 years ago, provides an important lesson for the analysis of the eukaryotic cell cycle. Before this experiment, temperature shifts had been used to synchronize bacteria and determine the pattern of DNA synthesis during the bacterial division cycle. These experiments indicated that DNA replication occurred during a fraction of the division cycle with gaps before and after DNA synthesis, a pattern similar to the eukaryotic division cycle. The SBM experiment studied DNA replication during the division cycle by labeling an unperturbed culture with a short pulse of tritiated thymidine. All cells were found to be labeled, indicating that unperturbed cells synthesize DNA throughout the division cycle. Thus, the SBM experiment was a control experiment demonstrating that artifacts can be introduced by synchronization methods. The idea of an control experiment under unperturbed conditions is proposed for the analysis of data on cell-cycle-specific gene expression in yeast and mammalian cells.

Artifacts↗

PrfA, the transcriptional activator of virulence genes, is upregulated during interaction of Listeria monocytogenes with mammalian cells and in eukaryotic cell extracts.

Most virulence genes of Listeria monocytogenes are activated by the transcriptional regulator PrfA. Previous studies have shown that environmental parameters, such as temperature, pH, stress conditions and medium composition, affect the expression of PrfA and PrfA-dependent proteins. In this report, we demonstrate a threefold increase in PrfA protein synthesis during infection of mammalian cells, which correlates with the increased activity of the plcA promoter, the major prfA promoter. Increased PrfA synthesis begins when L. monocytogenes adheres to host cells. In addition, we show that the observed induction of PrfA during the interaction of L. monocytogenes with mammalian cells can be reproduced in vitro using total cell extracts. Our data suggest a role for host proteinase K-sensitive protein(s) in PrfA upregulation.

Animals↗

GroEL heat shock protein of Haemophilus ducreyi: association with cell surface and capacity to bind to eukaryotic cells.

The Haemophilus ducreyi homolog of GroEL, a 58.5-kDa heat shock protein (Hsp), is a dominant protein produced not only in response to heat stress but also under in vitro growth conditions. Extracellular localization of the 58.5-kDa Hsp was investigated by whole-cell enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy and in supernatants of washed bacteria by immunoblotting with a Haemophilus ducreyi GroEL-specific mouse monoclonal antibody (BB11). To investigate binding of the Hsp to eukaryotic cells, the 58.5-kDa Hsp was purified by ion-exchange and size exclusion chromatography; incubated with HEp-2 cells, HeLa cells, and human fibroblasts; and then analyzed by immunoblotting. Direct involvement of the 58.5-kDa Hsp in the adherence of H. ducreyi to HEp-2 cells was investigated by using an inhibition assay. An epitope of the 58.5-kDa Hsp was detected by whole-cell ELISA on all of the strains tested, suggesting that it is associated with the cell surface. This was also supported by immunoelectron microscopy results. In supernatants of washed bacteria, the 58.5-kDa Hsp was detected by immunoblotting after 10 h of cultivation. The 58.5-kDa Hsp bound to the eukaryotic cells tested but exerted only limited (about 20%) inhibition of H. ducreyi adherence to HEp-2 cells. These results demonstrate that the 58.5-kDa Hsp of H. ducreyi is associated with the bacterial surface, binds to eukaryotic cells, and partially influences H. ducreyi adherence to HEp-2 cells, indicating possible involvement of the 58.5-kDa Hsp in the attachment of bacteria to host cells and to each other.

Animals↗

Isoprene synthesis in isolated embryonic Drosophila cells. I. Sterol-deficient eukaryotic cells.

Since insects are cholesterol auxotrophs, we analyzed the apparent paradox presented by an established cell line (Kc cells) from Drosophila embryos which grew in media which contained less than 0.05 micrograms/ml of sterols. Fresh Drosophila embryos contained 3.7 micrograms of 3 beta-hydroxysterols/mg of protein; however, Kc cells had maximally 0.50 micrograms of 3 beta-hydroxysterols/mg of protein. Kc cells, grown in media which contained cholesterol, showed the presence of sterol in their plasma and intracellular membranes. Kc cells did not synthesize sterols or any apparent replacement lipophilic molecule. However, two major compounds which comigrated with ubiquinone and dolichol were synthesized from radioactive mevalonate and acetate. Cholesterol incorporation into Kc cell membranes did not significantly alter total phospholipid head or acyl group composition. Similar observations were obtained with Schneider's Drosophila cell line I and a mosquito (Aedes albopictus) cell line. Our results (a) challenged current concepts that sterols or related replacement isopentenoid molecules were required for eukaryotic membrane structure, (b) demonstrated that marked alterations in eukaryotic membrane sterol composition was insufficient to change total phospholipid head and/or acyl group composition, and (c) set the stage for the use of a eukaryotic cell to examine the regulation of 3-hydroxy,3-methylglutaryl coenzyme A reductase activity independent of a requirement for sterol synthesis.

Animals↗