Biomedical subjects

Grzegorz Wegrzyn

Publications and source records attributed to Grzegorz Wegrzyn.

At least 19 recordsLinked to original sources

PubMed2006

Adenosine monophosphate-induced amplification of ColE1 plasmid DNA in Escherichia coli.

ColE1 plasmid copy number was analyzed in relaxed (relA) and stringent (relA(+)) Escherichia coli cells after supplementation of culture media with adenosine monophosphate (AMP). When a relaxed E. coli strain bearing ColE1 plasmid was cultured in LB medium for 18 h and induced with AMP for 4h, the plasmid DNA yield was significantly increased, from 2.6 to 16.4 mgl(-1). However no AMP-induced amplification of ColE1 plasmid DNA was observed in the stringent host. Some plasmid amplification was observed in relA mutant cultures in the presence of adenosine, while adenine, ADP, ATP, ribose, potassium pyrophosphate and sodium phosphate caused a minor, if any, increase in ColE1 copy number. A mechanism for amplification of ColE1 plasmid DNA with AMP in relA mutant bacteria is suggested, in which AMP interferes with the aminoacylation of tRNAs, increases the abundance of uncharged tRNAs, and uncharged tRNAs promote plasmid DNA replication. According to this proposal, in relA(+) cells, the AMP induction could not increase ColE1 plasmid copy number because of lower abundance of uncharged tRNAs. Our results suggest that the induction with AMP can be used as an effective method of amplification of ColE1 plasmid DNA in relaxed strains of E. coli.

Adenosine Monophosphate↗

PubMed2006

Effects of the presence of ColE1 plasmid DNA in Escherichia coli on the host cell metabolism.

BACKGROUND: Although understanding of physiological interactions between plasmid DNA and its host is important for vector design and host optimization in many biotechnological applications, to our knowledge, global studies on plasmid-host interactions have not been performed to date even for well-characterized plasmids. RESULTS: Escherichia coli cells, either devoid of plasmid DNA or bearing plasmid pOri1 (with a single ColE1 replication origin) or plasmid pOri2 (with double ColE1 replication origins), were cultured in a chemostat. We used a combination of metabolic flux analysis, DNA microarray and enzyme activity analysis methods to explore differences in the metabolism between these strains. We found that the presence of plasmids significantly influenced various metabolic pathways in the host cells, e.g. glycolysis, the tricarboxylic acid (TCA) cycle and the pentose phosphate (PP) pathway. Expression of rpiA, a gene coding for ribose-5-phosphate isomerase A, was considerably decreased in E. coli carrying a high copy number plasmid relative to E. coli carrying a low copy number plasmid and plasmid-free E. coli. The rpiA gene was cloned into an expression vector to construct plasmid pETrpiA. Following induction of pETrpiA-bearing E. coli, which harbored either pOri1 or pOri2, with isopropyl-beta-D-thiogalactopyranoside (IPTG), the copy number of pOri1 and pOri2 was sigificantly higher than that measured in a host devoid of pETrpiA. CONCLUSION: The presence of plasmids can significantly influence some metabolic pathways in the host cell. We believe that the results of detailed metabolic analysis may be useful in optimizing host strains, vectors and cultivation conditions for various biotechnological purposes.

Journal Article↗

PubMed2006

Direct addition of cultures of tester bacteria into semi-permeable membrane devices (SPMDs) as a modified procedure for preliminary detection of mutagenic pollution of the marine environment by use of microbiological mutagenicity assays.

Mutagenic pollution of the natural environment, including marine waters, is a very serious ecological problem. However, since chemical mutagens usually occur and act at low concentrations, their detection and identification is technically difficult, laborious and time-consuming. Therefore, preliminary detection of mutagenic pollution is commonly based on biological mutagenicity assays. On the other hand, triolein-containing semi-permeable membrane devices (SPMDs) provide a method for concentration of hydrophobic organic contaminants, including a large fraction of the mutagens. Combinations of SPMDs with microbiological toxicity and mutagenicity assays have already been described, but only SPMD-derived extracts, prepared with various organic solvents, were tested in such a way to date. We found that the presence of these solvents could interfere with the Vibrio harveyi bioluminescence-based mutagenicity assay. Moreover, preparation of the extracts from SPMD takes usually at least 48h. Here, we propose a modified procedure, based on direct addition of tester bacteria cultures into SPMD. We found that this procedure is significantly (at least two times) more rapid and several times more sensitive than that based on testing the extracts. This optimization is presented in this report. Moreover, we have performed preliminary studies on samples of marine waters. Positive results (i.e. detection of mutagenic activity) were obtained when test samples came from a region known to be highly contaminated by industrial pollution, while negative results were observed in the case of samples from a region supposed to be of low risk for mutagenic pollution.

Bacteria↗

PubMed2006

Genistein-mediated inhibition of glycosaminoglycan synthesis as a basis for gene expression-targeted isoflavone therapy for mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) are inherited, severe, progressive, metabolic disorders caused by deficiencies in different enzymes involved in degradation of glycosaminoglycans (GAGs). Although enzyme replacement therapy (ERT) has recently been available for MPS type I, and clinical trials have been performed in ERT for MPS II and MPS VI, there is little chance that this kind of treatment may be effective for neurodegenerative forms of MPS (due to inefficient delivery of enzymes to central nervous system through the blood-brain barrier), hence currently there is no effective therapy available for them. Therefore, we aim to develop an alternative therapy for these diseases. We found that genistein (4',5,7-trihydroxyisoflavone or 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) inhibits synthesis of GAGs considerably in cultures of fibroblasts of MPS patients (types I, II, IIIA and IIIB were tested). Prolonged cultivation of these cells in the presence of genistein resulted in reduction of GAG accumulation and normalization of cells as estimated by biochemical tests and electron microscopic analysis, respectively. As genistein inhibits kinase activity of epidermal growth factor receptor, which is required for full expression of genes coding for enzymes involved in GAG production, we propose to consider a substrate reduction therapy for MPS, which is referred to as 'gene expression-targeted isoflavone therapy'.

Biopsy↗

PubMed2006

DNA replication defect in the Escherichia coli cgtA(ts) mutant arising from reduced DnaA levels.

In Escherichia coli and other bacteria, the ribosome-associated CgtA GTP-binding protein plays a critical role in many basic cellular processes, including the control of DNA replication and/or segregation. However, the mechanism of this control is largely unknown. Here we report that ectopic expression of the dnaA gene partially restored both early growth in liquid medium and DNA synthesis defects of the cgtA(ts) mutant. Amounts of DnaA protein in the cgtA(ts) mutant incubated at elevated (42 degrees C) temperature were significantly lower relative to wild-type bacteria. Both level of dnaA mRNA and transcriptional activity of the dnaA promoter-lacZ fusion were decreased in the CgtA-deficient cells. The effects of ectopic expression of dnaA were specific as analogous expression of another gene coding for a replication regulator, seqA, had no significant changes in growth and DNA synthesis in the cgtA mutant. Thus, it appears that the DNA replication defect in this mutant is a consequence of reduced DnaA levels.

Artificial Gene Fusion↗

PubMed2006

Mutagenic activity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a neurotoxin, which can damage dopaminergic neurons. It causes symptoms resembling those observed in patients suffering from Parkinson's disease, and hence this toxin is widely used in studies on animal models of this disorder. Mutagenicity of MPTP was also reported by some authors, but results obtained by others suggested that this compound is not mutagenic. Interestingly, those contrasting results were based on the same assay (the Ames test). Therefore, we aimed to test MPTP mutagenicity by employing a recently developed Vibrio harveyi assay, which was demonstrated previously to be more sensitive than the Ames test, at least for some mutagens. We found that MPTP showed a significant mutagenic activity. Moreover, MPTP mutagenicity was attenuated by methylxanthines, compounds that are known to form complexes with aromatic mutagens.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2006

Phosphorylation of Escherichia coli poly(A) polymerase I and effects of this modification on the enzyme activity.

In Escherichia coli, RNA polyadenylation is catalyzed mainly by poly(A) polymerase I (PAP I). Here we demonstrate that a PAP I variant with a C-terminal His tag (PAP I-His) can be phosphorylated both in vivo and in an artificial in vitro system. The in vivo phosphorylation of PAP I-His impairs activity of this enzyme. Previous studies, performed by others, indicated that phosphorylation of His-tagged proteins usually reflects such a modification of their native counterparts in bacterial cells. Therefore, our results suggest that phosphorylation and dephosphorylation of PAP I may be important regulatory processes in the control of activity of this enzyme.

Escherichia coli↗

PubMed2006

Expression of genes coding for GerA and GerK spore germination receptors is dependent on the protein phosphatase PrpE.

The ability of Bacillus subtilis to form spores is a strategy for survival under unfavorable environmental conditions. It is equally crucial to break spore dormancy and return to vegetative growth at the appropriate time. Here we present data showing that the PrpE phosphatase is involved in the control of expression of genes coding for GerA receptors, which are necessary for L-alanine-induced spore germination. Moreover, PrpE is also involved in aspartic acid, glucose, fructose, and potassium (AGFK)-induced spore germination by controlling expression of genes coding for GerK receptors. In the absence of PrpE, the production of spores was essentially normal. However, L-alanine-induced spore germination and, to a lesser extent, the AGFK-induced pathway were abolished. In contrast, the germination pathway dependent on Ca2+-dipicolinate or dodecylamine remained intact. A protein phosphatase PrpE-green fluorescent protein fusion was localized to the prespore and to the dormant spore, consistent with a role in controlling expression of genes coding for GerA receptors. We propose that PrpE is an important element in a signal transduction pathway in Bacillus subtilis that controls the expression of genes coding for germination receptors.

Bacillus subtilis↗

PubMed2006

[Bacteriophage lambda DNA replication--new discoveries made using an old experimental model].

Bacteriophage lamda is a model in molecular biology studies since over fifty years. Nevertheless, studies of recent years (similarly to previous time periods) resulted in many new experimental results which not only expanded our knowledge on molecular mechanisms of functions of this virus, but also shed new light on general rules of the transduction and transfer of genetic information. In this review, we present recent achievements of studies on mechanisms of regulation of bacteriophage lamda DNA replication. Between others, these studies led to determination of the composition of lamda inherited replication complex, indication of the biological role of rapid degradation of free lamda omicron protein, description of the proposal of regulation of the switch from early (theta) to late (sigma) lamda DNA replication mode, elucidation of the mechanism of transcription regulation by a replication protein DnaA and demonstration of the activity of transcription stimulator by another replication regulator--the SeqA protein. These results may be important to better understand regulation of DNA replication of not only bacteriophage lamda but also other organisms.

Bacterial Proteins↗

PubMed2005

Changes in hair morphology of mucopolysaccharidosis I patients treated with recombinant human alpha-L-iduronidase (laronidase, Aldurazyme).

Mucopolysaccharidoses (MPS) are heritable, metabolic diseases caused by accumulation of mucopolysaccharides (glycosaminoglycans, GAGs) in lysosomes. This accumulation is due to a deficiency in one of several specific enzymes involved in the degradation of GAGs. MPS type I (MPS I) is caused by low or undetectable activity of alpha-L-iduronidase, an enzyme involved in removing the terminal iduronic acid residues from heparan and dermatan sulfate. Recently, an enzyme replacement therapy (ERT) for MPS I, based on administration of recombinant human alpha-L-iduronidase (laronidase, Aldurazyme), became available. The assessment of efficacy of ERT is especially important because MPS I is a highly variable and very rare disease, and the clinical trials involved relatively low number of patients. Among various significant clinical improvements during ERT, remarkable changes in hair morphology were noted. Detailed studies of hair samples from one patient, who did not have a hair cut from the beginning of ERT to the end of this study, and supported by results obtained for two other patients, revealed hair shaft structural abnormalities in MPS I hair. These hair abnormalities disappeared upon treatment with Aldurazyme. Although hair morphology is of limited clinical importance, the data suggest that changes in this parameter could be a useful, additional tool for a rapid, non-invasive, preliminary assessment of ERT efficacy.

Adolescent↗

PubMed2005

Differential antibacterial activity of genistein arising from global inhibition of DNA, RNA and protein synthesis in some bacterial strains.

Antibacterial activities of various flavonoids have been reported previously, but mechanism(s) of their action on bacterial cells remain(s) largely unknown. Here, we investigated effects of genistein, an isoflavone, and representatives of other flavonoids: daidzein (another isoflavone), apigenin (a flavone), naringenin (a flavanone) and kaempferol (a flavonol), on commonly used laboratory strains of model bacterial species: Escherichia coli, Vibrio harveyi and Bacillus subtilis. We found that E. coli was resistant to all tested flavonoids at concentrations up to 0.1 mM, while high sensitivity of V. harveyi to most of them (except daidzein, which exhibited significantly less pronounced effect) was observed. Effects of the flavonoids on B. subtilis were relatively intermediate to the two extremes, i.e., E. coli and V. harveyi. Action of genistein on bacterial cells was investigated in more detail to indicate changed cell morphology (formation of filamentous cells) of V. harveyi and drastic inhibition of global synthesis of DNA and RNA as shortly as 15 min after addition of this isoflavone to a bacterial culture to a final concentration of 0.1 mM. Protein synthesis inhibition was also apparent, but delayed. Both cell morphology and synthesis of nucleic acids and proteins were unaffected in E. coli cultures under analogous conditions. Studies on cell survival suggest that genistein is a bacteriostatic agent rather than a bactericidal compound.

Anti-Bacterial Agents↗

PubMed2005

Transcription start sites in the promoter region of the Escherichia coli pcnB (plasmid copy number) gene coding for poly(A) polymerase I.

Escherichia coli pcnB gene was discovered as a regulator of ColE1 plasmid copy number about 20 years ago. Further studies indicated that this gene codes for poly(A) polymerase I (PAP I). However, although importance of RNA polyadenylation has been demonstrated and biochemical properties of PAP I have been studied extensively, little is known about regulation of the pcnB gene transcription. Here, we demonstrate that there are three transcription start sites upstream of the pcnB coding sequence. Activities of at least two of these promoters were in inverse correlation to bacterial growth rates, which agrees with recently published results indicating increased abundance of PAP I and higher levels of RNA polyadenylation in slowly growing bacteria.

Base Sequence↗

PubMed2005

Transcription in the prpC-yloQ region in Bacillus subtilis.

The prpC and prkC genes encode two proteins with opposing activities, eukaryotic-like protein phosphatase and protein kinase involved in the sporulation and biofilm formation processes. The prpC gene overlaps 3 bp of the prkC gene; prkC is followed by the essential gene, yloQ. The organisation of the prpC, prkC and yloQ genes is conserved among several Gram-positive bacteria. In this work, we have found two promoters which function within the region of these genes and we also described conditions in which these promoters become activated. One promoter appears to be located upstream of prpC resulting in transcription of both prpC and prkC, as well as the yloQ gene. A second promoter is located 7 bp upstream of the start codon of yloQ. The yloQ promoter was activated during ethanol stress, but was sigmaB-independent. We also showed that prpC, prkC and yloQ genes form an operon.

Bacillus subtilis↗

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

A bacterial model for studying effects of human mutations in vivo: Escherichia coli strains mimicking a common polymorphism in the human MTHFR gene.

A simple bacterial model for studying effects of human mutations in vivo, when homologous genes exist in bacterial and human cells, is presented. We have constructed Escherichia coli strains bearing different alleles of the metF gene, an ortologue of human MTHFR gene, coding for 5,10-methylenetetrahydrofolate reductase. These strains bear a null mutation in the chromosomal metF gene and different metF alleles on plasmid(s), and thus there are merozygotes mimicking wild-type homozygotes, heterozygotes and recessive mutant homozygotes. The A177V mutantion in metF corresponds to one of the most common MTHFR polymorphism, A222V, which has been shown to be associated with increased levels of homocysteine in plasma that, in turn, causes many serious medical problems. Results of relatively simple and quick experiments with these strains are compatible with previously published reports on effects of the A222V substitution in the product of MTHFR gene. In addition, these results suggest either impairment of formation of heterodimers and/or heterotetramers by wild-type and A177V metF variants or dominance of the wild-type polypepides in such structures. Moreover, positive effects of folic acid and vitamins B2 and B12 on physiology of the mutant cells, suggested on the basis of clinical studies, is confirmed. Therefore, we conclude that the bacterial model described in this report may be a useful tool in studies on human mutations.

Bacterial Proteins↗

PubMed2005

Atypical microbial infections of digestive tract may contribute to diarrhea in mucopolysaccharidosis patients: a MPS I case study.

BACKGROUND: Mucopolysaccharidoses are heritable, metabolic diseases caused by deficiency in an activity of one of specific lysosomal enzymes involved in degradation of mucoplysaccharides (glycosaminoglycans). Among many medical problems of patients with mucopolysaccharidoses, there are frequent episodes of diarrhea of unknown etiology. CASE PRESENTATION: A girl, diagnosed enzymatically for mucopolysaccharidosis type I (deficiency of alpha-L-iduronidase) at the age of 3 years and 9 months, was investigated until the age of 5 years and 4 months. Frequent loose stools and episodes of diarrhea, often accompanied by vomiting, were encountered. Detailed microbiological analyses were performed and atypical microbial infections (most often enetropathogenic Escherichia coli, but also other species, like Pseudomonas aeruginosa or Staphylococcus aureus, as well as adenoviruses) of the digestive tract were found in most severe diarrhea episodes. Often, isolations of pathogenic bacterial strains from stools of the investigated patient suffering from diarrhea were not obvious during the first screening, and only detailed microbiological studies, including re-isolation of colonies, gave the results of isolation of particular pathogenic strains (especially in the case of enetropathogenic E. coli). CONCLUSION: We conclude that atypical microbial infections of digestive tract may contribute significantly to diarrhea in mucopolysaccaridosis patients. Since isolated strains were not typical and their isolation was often possible only after detailed investigation (not during a standard screening), such atypical microbial infections of digestive tract of mucopolysaccharidosis patients could be usually overlooked to date. Importantly, these atypical infections could be effectively treated with antimicrobial agents.

Adenoviridae Infections↗

PubMed2005

Localization of Escherichia coli poly(A) polymerase I in cellular membrane.

Poly(A) polymerase I (PAP I), the pcnB gene product, is the main enzyme responsible for RNA polyadenylation in Escherichia coli. Polyadenylated RNA molecules are rapidly degraded by a multiprotein complex called RNA degradosome. Here we demonstrate that apart from its presence in cytosol, PAP I is also localized in cellular membrane. Although this observation might appear surprising, it was demonstrated recently by others that E. coli RNA degradosome is also associated with the cytoplasmic membrane. Moreover, we show that development of single-stranded RNA bacteriophages MS2 and Qbeta, but not that of single-stranded DNA bacteriophage M13, is more efficient in the pcnB mutant relative to an otherwise isogenic pcnB(+) host.

Cell Membrane↗