Biomedical subjects

Agata Czyz

Publications and source records attributed to Agata Czyz.

15 recordsLinked to original sources

PubMed2005

Bioluminescence-mediated stimulation of photoreactivation in bacteria.

Although biochemistry and genetics of light emission by cells have been investigated in detail, a biological role for bacterial luminescence has remained obscure for a long time. It was proposed recently that luminescence may stimulate DNA repair, but the specific mechanism of this phenomenon was not investigated. Moreover, experiments showing decreased survival of UV-irradiated lux mutants relative to luminescent cells were performed previously using only one bacterial species, Vibrio harveyi. Here, we demonstrate that dark mutants of various strains of naturally luminescent bacteria (Photobacterium leiognathi, Photobacterium phosphoreum and Vibrio fischeri) are more sensitive to UV irradiation than wild-type cells. Thus, this phenomenon occurs not only in V. harveyi but also in other bacterial species. Using an artificial system of luminescent Escherichia coli in combination with phr mutants (defective in photolyase functions), we found that bacterial luminescence may stimulate photoreactivation, perhaps by providing photons that are necessary for photolyase activity.

Aliivibrio fischeri↗

PubMed2005

Methylxanthines (caffeine, pentoxifylline and theophylline) decrease the mutagenic effect of daunomycin, doxorubicin and mitoxantrone.

Previously performed experiments showed that methylxanthines, especially caffeine, may protect cells against cytostatic or cytotoxic effects of several aromatic compounds. One of the proposed mechanisms of this protection is based on stacking interactions between pi electron systems of polycyclic aromatic molecules. In this work, we demonstrate that caffeine and other methylxanthines--pentoxifylline and theophylline--significantly decrease mutagenicity of the anticancer aromatic drugs daunomycin, doxorubicin and mitoxantrone. The spectrophotometric titration of these aromatic compounds by methylxanthines indicated formation of mixed aggregates. The concentrations of free active forms of the drugs decreased when the concentrations of methylxanthines increased in the mixture. Therefore, likely methylxanthines may play a role of scavengers of the free active forms of daunomycin, doxorubicin and mitoxantrone.

Antimutagenic Agents↗

PubMed2005

The Obg subfamily of bacterial GTP-binding proteins: essential proteins of largely unknown functions that are evolutionarily conserved from bacteria to humans.

Members of the Obg subfamily of small GTP-binding proteins (called Obg, CgtA, ObgE or YhbZ in different bacterial species) have been found in various prokaryotic and eukaryotic organisms, ranging from bacteria to humans. Although serious changes in phenotypes are observed in mutant bacteria devoid of Obg or its homologues, specific roles of these GTP-binding proteins remain largely unknown. Recent genetic and biochemical studies, as well as determination of the structures of Obg proteins from Bacillus subtilis and Thermus thermophilus, shed new light on the possible functions of the members of the Obg subfamily and may constitute a starting point for the elucidation of their exact biological role.

Bacterial Proteins↗

PubMed2004

Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips.

Infections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories. A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here. Different phages of Escherichia coli and Bacillus subtilis were analyzed. Phage DNA was isolated from bacterial culture samples and detected by combination of bead-based sandwich hybridization with enzyme-labeled probes and detection of the enzymatic product using silicon chips. The assay resulted in specific signals from all four tested phages without significant background. Although high sensitivity was achieved in 4h assay time, a useful level of sensitivity (10(7)-10(8) phages) is achievable within 25 min. A multiplex DNA chip technique involving a mixture of probes allows for detection of various types of phages in one sample. These analyses confirmed the specificity of the assay.

Bacillus subtilis↗

PubMed2004

Bacteriophage contamination: is there a simple method to reduce its deleterious effects in laboratory cultures and biotechnological factories?

Infection of bacterial cultures by bacteriophages as well as prophage induction in the host cells are serious problems in both research and biotechnological laboratories. Generally, prevention strategies (like good laboratory/factory hygiene, sterilisation, decontamination and disinfection) are necessary to avoid bacteriophage contamination. However, it is well known that no matter how good the laboratory/factory practice and hygiene are, bacteriophage infections occur from time to time. The use of immunised or resistant bacterial strains against specific phages may be helpful, but properties of the genetically modified strains resistant to phages are often worse (from the point of view of a researcher or a biotechnological company) than those of the parental, phage-sensitive strains. In this article we review recent results that may provide a simple way to minimise deleterious effects of bacteriophage infection and prophage induction. It appears that low bacterial growth rates result in a significant inhibition of lytic development of various bacteriophages. Moreover, spontaneous prophage induction is less frequent in slowly growing bacteria.

Bacteria↗

PubMed2003

Alleviation of mutagenic effects of polycyclic aromatic agents (quinacrine mustard, ICR-191 and ICR-170) by caffeine and pentoxifylline.

Previous studies performed by others indicated that apart from its other biological effects, caffeine (CAF) may have a role in protection of organisms against cancer. However, biological mechanism of this phenomenon remained unknown. Recent studies suggested that caffeine can form stacking (pi-pi) complexes with polycyclic aromatic chemicals. Therefore, one might speculate that effective concentrations of polycyclic aromatic mutagens could be reduced in the presence of caffeine. Here we demonstrate that caffeine and another xanthine, pentoxifylline (PTX), effectively alleviate mutagenic action of polycyclic aromatic agents (exemplified by quinacrine mustard (QM), 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (ICR-191) and 1,3,7-propanediamine-N-(2-chloroethyl)-N'-(6-chloro-2-methoxy-9-acridinyl)-N-ethyl.2HCl (ICR-170)), but not of aliphatic mutagens (exemplified by mechlorethamine), in the recently developed mutagenicity test based on bacterium Vibrio harveyi. Biophysical studies indicated that caffeine and pentoxifylline can form stacking complexes with the aromatic agents mentioned above. Molecular modeling also confirmed a possibility of stacking interactions between examined molecules.

Aminacrine↗

PubMed2003

Experimental evidence for the physiological role of bacterial luciferase in the protection of cells against oxidative stress.

The origin and function of bioluminescence was considered a problematic question of the Charles Darwin theory. Early evolution of bacterial luminescence and its current physiological importance seem to be especially mysterious. Recently, it was proposed that stimulation of DNA repair may be a physiological role for production of light by bacterial cells. On the other hand, it was also proposed that primary role of luminescent systems could be detoxification of the deleterious oxygen derivatives. Although some previous results might suggest that this hypothesis can be correct, until now experimental evidence for such a mechanism operating in bacterial cells and having physiological importance was generally lacking. Here we demonstrate that in the presence of various oxidants (hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, and ferrous ions) at certain concentrations in the culture medium, growth of Vibrio harveyi mutants luxA and luxB, but not of the mutant luxD, is severely impaired relative to wild-type bacteria. This deleterious effect of oxidants on the mutants luxA and luxB could be significantly reduced by addition of the antioxidants A-TEMPO or 40H-TEMPO. We conclude that bacterial luciferase may indeed play a physiological role in the protection of cells against oxidative stress.

Antioxidants↗

PubMed2003

Role of the cgtA gene function in DNA replication of extrachromosomal elements in Escherichia coli.

The cgtA gene codes for a common GTP-binding protein whose homologues were found in all prokaryotic and eukaryotic organisms investigated so far. Although cgtA is an essential gene in most bacterial species, its precise functions in the regulation of cellular processes are largely unknown. In Escherichia coli, dysfunction or overexpression of the cgtA gene causes problems in various chromosomal functions, like synchronization of DNA replication initiation and partitioning of daughter chromosomes after a replication round. It is not know how the cgtA gene product regulates these processes. Here we investigated effects of cgtA dysfunction on replication of plasmid and phage replicons. We found that replication of some plasmids (e.g., ColE1-like) is not affected in the cgtA mutant. On the other hand, dysfunction of the cgtA gene caused a strong inhibition of lambda plasmid DNA replication. Bacteriophage lambda development was severely impaired in the cgtA mutant. Replication of other plasmid replicons (derivatives of F, R1, R6K, and RK2) was influenced by the cgtA mutation moderately. It seems that DNA synthesis per se is not affected by CgtA, and that this protein might control replication initiation indirectly, by regulation of function(s) or production of one or more replication factors. In fact, we found that level of the host-encoded replication protein DnaA is significantly decreased in the cgtA mutant. This indicates that CgtA is involved in the regulation of dnaA gene expression.

Bacterial Proteins↗

PubMed2002

Comparison of the Ames test and a newly developed assay for detection of mutagenic pollution of marine environments.

A new assay for detection of mutagenic pollution of marine environments, based on the use of a series of genetically modified Vibrio harveyi strains, was developed recently. Here, we compared this assay with a commonly used Ames test, employing mutants of Salmonella enterica serovar Typhimurium. We found that survival of S. enterica serovar Typhimurium in artificial marine water and in different samples of marine water is dramatically reduced relative to that of V. harveyi strains. This indicates that V. harveyi strains are significantly more useful in testing samples of marine water. Moreover, sensitivity of both assays was compared. We found that using the V. harveyi assay it is possible to detect significantly (from a few to several times) lower concentrations of typical chemical mutagens than employing the Ames test. Although the higher sensitivity of the V. harveyi test relative to the Ames assay may be of minor importance when testing mutagenicity of certain chemicals in their pure form under laboratory conditions, this feature is very important in testing samples of marine water. Therefore, we assume that the V. harveyi assay may be an adequate test for detection of mutagenic pollution of marine environments.

Environmental Monitoring↗

PubMed2002

A role for the common GTP-binding protein in coupling of chromosome replication to cell growth and cell division.

Homologues of CgtA, the common GTP-binding protein of Vibrio harveyi, are present in diverse organisms ranging from bacteria to humans. In bacteria, proteins homologous to CgtA form a subfamily of small GTP-binding proteins, called Obg/Gtp1. Similarity between bacterial members of this subfamily and their eukaryotic homologues is as high as about 50%. Nevertheless, specific functions of these proteins remain largely unknown. Genes coding for CgtA-like proteins are essential in almost all species of bacteria. The only known exception is V. harveyi, whose cells survive disruption of the cgtA gene. Therefore, the V. harveyi cgtA insertional mutant is a very useful tool for studies on functions of CgtA. Here we demonstrate that under normal growth conditions, cells of the cgtA mutant are slightly larger than wild-type cells, whereas indirect inhibition of DNA replication initiation by addition of rifampicin results in significantly higher differences in average cell size between these two strains as measured by flow cytometry. These differences decreased when cell division was inhibited by cephalexin. DNA synthesis per cell mass was found to be increased in the cgtA mutant relative to wild-type V. harveyi strain, whereas the mutant cells grew slower than bacteria with functional cgtA gene. Kinetics of DNA replication after inhibition of cell division was also considerably different in wild-type and cgtA mutant strains. These results suggest that the cgtA gene product plays a role in coupling of DNA replication to cell growth and cell division.

Bacterial Proteins↗

PubMed2002

Impaired chromosome partitioning and synchronization of DNA replication initiation in an insertional mutant in the Vibrio harveyi cgtA gene coding for a common GTP-binding protein.

The Vibrio harveyi cgtA gene product belongs to a subfamily of small GTP-binding proteins, called Obg-like proteins. Members of this subfamily are present in diverse organisms ranging from bacteria to humans. On the other hand, the functions of these proteins in the regulation of cellular processes are largely unknown. Genes coding for these proteins are essential in almost all bacteria investigated thus far. However, a viable V. harveyi insertional mutant in the cgtA gene was described recently. Therefore, this mutant gives a unique opportunity to study functions of a member of the subfamily of Obg-like proteins. Here we demonstrate that the mutant cells often form long filaments with expanded, non-partitioned or rarely partitioned chromosomes. Such a phenotype suggests impairment of the mechanism of chromosome partition. Flow cytometric studies revealed that synchronization of chromosome replication initiation is also significantly disturbed in the cgtA mutant. Moreover, in contrast to wild-type V. harveyi, inhibition of chromosome replication and/or of cell division in the mutant bacteria caused significant increase in the number of large cells, suggesting that the cgtA gene product may be involved in the coupling of cell growth to chromosome replication and cell division. These results indicate that CgtA, an Obg-like GTP-binding protein, plays an important role in the regulation of chromosomal functions.

Bacterial Proteins↗

PubMed2002

Overexpression of the cgtA (yhbZ, obgE) gene, coding for an essential GTP-binding protein, impairs the regulation of chromosomal functions in Escherichia coli.

GTPases belonging to the Obg/Gtp1 subfamily are essential proteins in most bacterial species and are evolutionarily conservative from bacteria to humans. However, their specific functions in the regulation of cellular processes are largely unknown. Here we demonstrate that overproduction of a member of the Obg/Gtp1 subfamily, cgtA ( yhbZ, obgE) gene product, in Escherichia coli is deleterious for bacterial growth. However, syntheses of DNA, RNA, and proteins were not significantly affected under these conditions as measured by efficiency of incorporation of radioactive precursors. On the other hand, flow cytometry studies revealed that cgtA-overexpressing bacteria form enlarged cells with significantly changed distribution of chromosomal DNA. These results strongly suggest that overproduction of a GTP-binding protein from the Obg/Gtp1 subfamily impairs regulation of some chromosomal functions in E. coli, especially synchronization of DNA replication initiation and possibly also partitioning of daughter chromosomes after a replication round.

Bacterial Proteins↗

PubMed2002

Induction of light emission by luminescent bacteria treated with UV light and chemical mutagens.

Intensity of light emission by luminescent bacteria in response to UV irradiation and chemical mutagens was tested. We demonstrated that luminescence of six strains of marine bacteria (belonging to four species: Photobacterium leiognathi, P. phosphoreum, Vibrio fischeri and V. harveyi) is significantly increased by UV irradiation relatively shortly after dilution of cultures. Such a stimulation of luminescence was abolished in cells treated with chloramphenicol 15 min before UV irradiation, indicating that effective gene expression is necessary for UV-mediated induction of light emission. These results suggest that stimulation of luminescence in UV-irradiated bacterial cells may operate independently of the quorum sensing regulation. A significant induction of luminescence was also observed upon treatment of diluted cultures of all investigated strains with chemical mutagens: sodium azide (SA), 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine x 2HCl (ICR-191), 4-nitro-o-phenylenediamine (NPD), 4-nitroquinolone-N-oxide (NQNO), 2-aminofluorene (2-AF), and benzo[alpha]pyrene. These results support the proposal that genes involved in bioluminescence belong to the SOS regulon. The use of bacterial luminescence systems in assays for detection of mutagenic compounds is discussed in the light of this proposal.

Bacterial Proteins↗

PubMed2000

Vibrio harveyi bioluminescence plays a role in stimulation of DNA repair.

Although the genetics and biochemistry of bacterial luminescence have been investigated extensively, the biological role of this phenomenon remains unclear. Here it is shown that luxA, luxB and luxD mutants (unable to emit light) of the marine bacterium Vibrio harveyi are significantly more sensitive to UV irradiation when cultivated in the dark after irradiation than when cultivated under a white fluorescent lamp. This difference was much less pronounced in the wild-type (luminescent) V. harveyi strain. Survival of UV-irradiated Escherichia coli wild-type cells depended on subsequent cultivation conditions (in the dark or in the presence of external light). However, after UV irradiation, the percentage of surviving E. coli cells that bear V. harveyi genes responsible for luminescence was significantly higher than that of non-luminescent E. coli, irrespective of the subsequent cultivation conditions. Moreover, it is demonstrated that luminescence of V. harveyi can be stimulated by UV irradiation even in diluted cultures, under conditions when light emission by these bacteria is normally impaired due to quorum sensing regulation. It is proposed that luminescent bacteria have an internal source of light which could be used in DNA repair by a photoreactivation process. Therefore, production of internal light ensuring effective DNA repair seems to be at least one of the biological functions of bacterial luminescence.

Bacterial Proteins↗

PubMedDate not supplied

Stimulation of DNA repair as an evolutionary drive for bacterial luminescence.

It was demonstrated recently that luminescence of a free-living marine bacterium, Vibrio harveyi, stimulates DNA repair, most probably by activation of the photoreactivation process. Here, we ask whether the stimulation of DNA repair could be an evolutionary drive that ensured maintenance and development of early bacterial luminescent systems. To test this hypothesis, we cultivated V. harveyi lux(+) bacteria and luxA mutants in mixed cultures. Initial cultures were mixed to obtain a culture consisting of roughly 50% lux(+) cells and 50% luxA mutants. Then bacteria were cultivated for several days and ratio of luminescent to dark bacteria was measured. Under these conditions, luxA mutants became highly predominant within a few days of cultivation. This indicates that, without a selective pressure, the luminescence is a disadvantage for bacteria, perhaps due to consumption of significant portion of cell energy. However, when the same experiments were repeated but cultures were irradiated with low UV doses, luminescent bacteria started to predominate shortly after the irradiation. Therefore, we conclude that stimulation of photoreactivation may be an evolutionary drive for bacterial bioluminescence.

DNA Repair↗