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At least 19 recordsLinked to original sources
PubMed1997

Eukaryotic and prokaryotic cell functions required for invasion of Staphylococcus aureus into bovine mammary epithelial cells.

Eukaryotic and prokaryotic cellular functions required for invasion of Staphylococcus aureus into bovine mammary epithelial cells were investigated. Two strains of S. aureus isolated from milk of cows with clinical mastitis, a primary bovine mammary epithelial cell culture and a bovine mammary epithelial cell line were pretreated with inhibitors of nucleic acid and protein synthesis. In addition, mammary epithelial cells were pretreated with inhibitors of receptor-mediated endocytosis and oxidative phosphorylation. Protein and nucleic acid synthesis in prokaryotic and eukaryotic cells and eukaryotic oxidative phosphorylation were required for invasion of S. aureus into mammary epithelial cells. Inhibition of receptor-mediated endocytosis caused a significant reduction in the number of invading S. aureus. These results suggest that invasion of S. aureus into bovine mammary epithelial cells occurs through a receptor-mediated endocytosis process. Furthermore, eukaryotic oxidative metabolism, protein synthesis and nucleic acid synthesis as well as bacterial protein synthesis are required for bacterial invasion.

Animals↗
PubMed1992

Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes.

A method to microscopically detect and identify individual cells of members of the domains Bacteria and Archaea is presented. rRNA-targeted oligonucleotides were 5' end labeled with the enzyme horseradish peroxidase and used for whole-cell hybridization. Specifically bound probe was visualized by the enzymatic formation of an intracellular precipitate from the substrate diaminobenzidine. Permeation of the enzyme-labeled probe into whole fixed cells of gram-negative bacteria required their pretreatment with lysozyme-EDTA, whereas permeability of some archaebacterial cells was improved by addition of detergent to the hybridization buffer. Hitherto we had not achieved penetration of enzyme-labeled probe into gram-positive bacteria and yeast cells. This method should be a valuable tool for identification of suitable prokaryotic cells in environments with elevated background fluorescence or in situations in which an epifluorescence microscope is not available.

Archaea↗
PubMed1993

Biomass growth rate during the prokaryote cell cycle.

The rate of biomass growth throughout the cell cycle of prokaryotes is important in the study of global regulation. Two limiting cases have generally been considered: the exponential model and the linear model. The exponential model is a logical expectation because protein, the main component of biomass of a bacterial cell, increases continuously during the cell cycle and therefore the means for synthesis of other cell components and metabolites also increases. In addition, during the cell cycle, ribosomes, the means of production of proteins, increase monotonically. As a consequence, the increase of all should be autocatalytic and the content of cell substance should be an exponential function of time. Two cellular components would not be expected to increase exponentially: the DNA and the cell envelope. The former because of the intermittent synthesis of the chromosome, and the latter because of changes in the surface-to-volume ratio with growth and division. In contrast to the exponential model, the linear model of Kubitschek postulates that the cell only increases its membrane transport capability over a brief period during the cell cycle, and, thus limited by transport, all cell components can increase only at a constant linear rate during most of the cell cycle. Other proposed models are intermediate and assume that the growth rate of the cell depends on some cell cycle event, such as the initiation of chromosome replication. The models have relevance to prokaryotes undergoing balanced growth; they may not be relevant to eukaryotic microbes or to eukaryotic cells in tissue culture that have endogenous rhythms or are controlled by protein growth factors. Logically, the models could possibly apply to a free-living cell that does not respond to environmental cues. Even under rigidly constant conditions, however, cells may try to respond to a stimulus that was periodic or regulatory under natural conditions, but is present at a constant level under the experimental culture condition. There are four classes of experiments that have been used to measure the accumulation of dry biomass or its components during the cell cycle of a bacterium, as typified by Escherichia coli. For the first class of experiments, the dimensions of living cells are measured under the microscope. So far, the experiments have been limited by the resolving power of the phase microscope, but adequate resolution should be possible with the confocal scanning light microscope or various video computer systems. Such experiments are called integral because augmentation of cell constituents is followed. The second class involves pulse-chase labeling of cells and then their separation into different phases of the cycle or age groups and measurement of the radioactivity per cell in the fractions. Such experiments are called differential in that the rate is measured directly instead of being deduced by comparing the total size at different times.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Cycle↗
PubMed1988

Animal viruses are able to fuse with prokaryotic cells. Fusion between Sendai or influenza virions and Mycoplasma.

Sendai and influenza virions are able to fuse with mycoplasmata. Virus-Mycoplasma fusion was demonstrated by the use of fluorescently labeled intact virions and fluorescence dequenching, as well as by electron microscopy. A high degree of fusion was observed upon incubation of both virions with Mycoplasma gallisepticum or Mycoplasma capricolum. Significantly less virus-cell fusion was observed with Acholeplasma laidlawii, whose membrane contains relatively low amounts of cholesterol. The requirement of cholesterol for allowing virus-Mycoplasma fusion was also demonstrated by showing that a low degree of fusion was obtained with M. capricolum, whose cholesterol content was decreased by modifying its growth medium. Fluorescence dequenching was not observed by incubating unfusogenic virions with mycoplasmata. Sendai virions were rendered nonfusogenic by treatment with trypsin, phenylmethylsulfonyl fluoride, or dithiothreitol, whereas influenza virions were made nonfusogenic by treatment with glutaraldehyde, ammonium hydroxide, high temperatures, or incubation at low pH. Practically no fusion was observed using influenza virions bearing uncleaved hemagglutinin. Trypsinization of influenza virions bearing uncleaved hemagglutinin greatly stimulated their ability to fuse with Mycoplasma cells. Similarly to intact virus particles, also reconstituted virus envelopes, bearing the two viral glycoproteins, fused with M. capricolum. However, membrane vesicles, bearing only the viral binding (HN) or fusion (F) glycoproteins, failed to fuse with mycoplasmata. Fusion between animal enveloped virions and prokaryotic cells was thus demonstrated.

Acholeplasma laidlawii↗
PubMed1992

An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins.

The functionally diverse actin, hexokinase, and hsp70 protein families have in common an ATPase domain of known three-dimensional structure. Optimal superposition of the three structures and alignment of many sequences in each of the three families has revealed a set of common conserved residues, distributed in five sequence motifs, which are involved in ATP binding and in a putative interdomain hinge. From the multiple sequence alignment in these motifs a pattern of amino acid properties required at each position is defined. The discriminatory power of the pattern is in part due to the use of several known three-dimensional structures and many sequences and in part to the "property" method of generalizing from observed amino acid frequencies to amino acid fitness at each sequence position. A sequence data base search with the pattern significantly matches sugar kinases, such as fuco-, glucono-, xylulo-, ribulo-, and glycerokinase, as well as the prokaryotic cell cycle proteins MreB, FtsA, and StbA. These are predicted to have subdomains with the same tertiary structure as the ATPase subdomains Ia and IIa of hexokinase, actin, and Hsc70, a very similar ATP binding pocket, and the capacity for interdomain hinge motion accompanying functional state changes. A common evolutionary origin for all of the proteins in this class is proposed.

Actins↗
PubMedDate not supplied

[Characteristics of the stoichiometric regulation of glycolysis in prokaryotic cells. A model].

A model is developed to investigate the steady state of the glycolytic system of procaryotic cells which have the hexose transport into the cell coupled with phosphoenolpyruvic acid-dependent phosphorylation. Analysis of the model shows that this phosphorylation results: 1) in stoichiometric connection between the rate of hexose consumption and the utilization of ATP and 2) the stoichiometric positive feedback. The latter can be the cause of bistability, oscillations and other nonlinear phenomena.

Bacteria↗
PubMed1989

Evidence for a membrane-associated GTP-binding protein in Stigmatella aurantiaca, a prokaryotic cell.

Signal transducing G proteins are present in all eukaryotic cells, but they have not been found in prokaryotes so far. Myxobacteria, especially Stigmatella aurantiaca, are prokaryotic organisms able to exchange signals. Moreover, they exhibit an active phosphoinositide metabolism, whose intensity is dependent on the physiological state of the cell. Therefore G proteins potentially involved in the activation of phospholipid metabolism or any other event stimulated by external signals were looked for in S. aurantiaca membranes. Using a photoaffinity technique based on cross-linking of radioactive GTP to membrane-associated proteins under UV irradiation, only one major band in the range of 54 kDa was detected. This GTP-binding protein present specifically in membrane preparations binds also GDP, whereas it does not react with other nucleotides, such as ATP, UTP and CTP. The membrane-bound G protein of S. aurantiaca needs further characterization but could be homologous to G alpha subunits found in cytoplasmic membranes of eukaryotes.

Affinity Labels↗
PubMed1984

Occurrence of S-adenosylhomocysteine hydrolase in prokaryote cells. Characterization of the enzyme from Alcaligenes faecalis and role of the enzyme in the activated methyl cycle.

The occurrence of S-adenosylhomocysteine hydrolase (EC 3.3.1.1) was found in a variety of prokaryotes. These prokaryotes did not exhibit any activities of S-adenosylhomocysteine nucleosidase (EC 3.2.2.9) and S-ribosyl-homocysteine hydrolase (EC 3.3.1.3), which had been the generally accepted prokaryote enzymes for the regeneration of free homocysteine from S-adenosylhomocysteine in the activated methyl cycle. In these prokaryotes S-adenosylhomocysteine hydrolase was suggested to be the only enzyme functioning for the regeneration of free homocysteine by enzymological and immunochemical studies. S-Adenosylhomocysteine hydrolase was purified and crystallized from cells of a prokaryote, Alcaligenes faecalis. The purified enzyme was found to be homogeneous on ultracentrifugation and gel electrophoresis. Its relative molecular mass is approximately 280 000 and it is composed of six identical subunits with a Mr of approximately 48 000. The NH2-terminal and COOH-terminal amino acids are lysine and tyrosine respectively. The enzyme contains 6 mol NAD/mol. Some nucleosides, such as formycin A, nebularine, adenosine N1-oxide and so on, are able to substitute for adenosine yielding the corresponding S-nucleosidylhomocysteine congeners. Modification of the 5'-hydroxymethyl group in adenosine leads to the most potent inhibition of the thioether formation of homocysteine with adenosine. The enzyme from A. faecalis has some immunological similarities to other prokaryote S-adenosylhomocysteine hydrolases, but is different from the enzymes of animal sources.

Adenosylhomocysteinase↗
PubMed1993

RNA processing in prokaryotic cells.

RNA processing in Escherichia coli and some of its phages is reviewed here, with primary emphasis on rRNA and tRNA processing. Three enzymes, RNase III, RNase E and RNase P are responsible for most of the primary endonucleolytic RNA processing events. The first two are proteins, while RNase P is a ribozyme. These three enzymes have unique functions and in their absence, the cleavage events they catalyze are not performed. On the other hand a relatively large number of exonucleases participate in the trimming of the 3' ends of tRNA precursor molecules and they can substitute for each other. Primary processing is the first event that happens to the nascent RNA molecule, while in secondary RNA processing, the substrate is a product of a primary processing event. Although most RNA processing occurs in RNP particles, it seems that only in secondary RNA processing is the RNP particle required for the reaction. Bacteria and especially bacteriophages contain self-splicing introns which in cases were probably acquired from other species.

Bacterial Proteins↗
PubMedDate not supplied

Inhibition of prokaryotic cell growth by HIV1 Vpr.

We have cloned the nef, vif, vpr and vpu genes of HIV1 in the pGEX system to produce auxiliary proteins of HIV1 as N-terminal fusions with glutathione S-transferase (GST). Some GST proteins are difficult to obtain under standard conditions. The synthesis and solubility varied considerably from one protein to another. We investigated the reasons for the poor production of GST-Vpr, GST-Vpu and GST-Vif. Interestingly, using this GST prokaryotic model, we demonstrated that Vpr, which is known to block the cell cycle of mammalian and yeast cells at the G2 phase, is also bacteriostatic for Escherichia coli. The effect on E. coli was specific to Vpr, and was not linked to the expression of the other HIV1 proteins. This suggests that Vpr interferes with components of cell replication that are conserved from prokaryotes to eukaryotes. Thus, E. coli appears to be a convenient model system for studies on the function of Vpr.

Cell Division↗
PubMed1992

Characterization of the glycoprotein D gene products of equine herpesvirus 1 using a prokaryotic cell expression vector.

The gene encoding equine herpesvirus 1 (equine abortion virus; EHV-1) glycoprotein D was engineered into the prokaryotic vector pEX, and expressed as a beta-galactosidase fusion product, which was recognized by pooled equine sera and anti-EHV-1 rabbit sera. Antibodies raised against the EHV-1 gD fusion product identified strong bands in infected cells at 66 and 68 K and at 138 K in purified virus, thus characterizing the several forms of this major envelope glycoprotein which is an important candidate for inclusion in subunit vaccines.

Animals↗
PubMed1982

In vitro mutagenic activity of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) in eukaryotic and prokaryotic cells.

The in vitro mutagenic activity of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC), has been studied in bacteria and Chinese hamster cells with and without metabolic activation by rat liver microsomes. DTIC was found to be highly mutagenic in the two systems. It is noteworthy that DTIC in the prokaryotic systems did not require metabolic activation to be effective. By comparing the mutagenic activity on bacteria of DTIC and of its monomethyl-and hydroxy-methyl-derivatives (MIC and HMIC), it is evident that MIC and HMIC display a pattern of mutagenicity different from DTIC. It suggests that neither MIC nor HMIC are the direct responsible metabolites for the mutagenic activity of DTIC in bacteria.

Animals↗
PubMed1998

Intracellular immunization of prokaryotic cells against a bacteriotoxin.

Intracellularly expressed antibodies have been designed to bind and inactivate target molecules inside eukaryotic cells. Here we report that an antibody fragment can be used to probe the periplasmic localization of the colicin A N-terminal domain. Colicins form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, they bind to a receptor located on the outer membrane and then are translocated through the envelope. The N-terminal domain of colicins is involved in the translocation step and therefore is thought to interact with proteins of the translocation system. To compete with this system, a single-chain variable fragment (scFv) directed against the N-terminal domain of the colicin A was synthesized and exported into the periplasmic space of E. coli. The periplasmic scFv inhibited the lethal activity of colicin A and had no effect on the lethal activity of other colicins. Moreover, the scFv was able to specifically inactivate hybrid colicins possessing the colicin A N-terminal domain without affecting their receptor binding. Hence, the periplasmic scFv prevents the translocation of colicin A and probably its interaction with import machinery. This indicates that the N-terminal domain of the toxin is accessible in the periplasm. Moreover, we show that production of antibody fragments to interfere with a biological function can be applied to prokaryotic systems.

Antibodies, Bacterial↗