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Biomedical subjects

E Grzesiuk

Publications and source records attributed to E Grzesiuk.

At least 19 recordsLinked to original sources
PubMed2006

DNA damage and its repair in lymphocytes and thyroid nodule cells during radioiodine therapy in patients with hyperthyroidism.

Radioiodine treatment of hyperthyroid patients with autonomous thyroid nodule leads to cellular DNA damage not only in thyrocytes but also in peripheral blood lymphocytes. The purpose of this study was to evaluate DNA breakage and base damage in thyrocytes and lymphocytes in patients treated with 131-I. In all the patients thyroid scintiscan was performed using 131-I. Damage to DNA was estimated by comet assay. Samples were taken before radioiodine treatment, and 12 and 54 days afterwards. Our results indicate high diversity in the level of DNA damage among the individual patients. However, in all cases, after 54 days the level of DNA damage in lymphocytes was similar or even lower than that in the controls. In contrast, in hot nodule the DNA damage persisted until the 54th day after 131-I application. Differences in the type of DNA damage between thyrocytes and lymphocytes were also observed. In lymphocytes there was more base damage, whereas in thyrocytes single strand breaks prevailed. This may indicate different mechanisms of DNA damage induction and/or DNA repair.

Adult↗
PubMed2006

Pancreatic secretion differs according to the genotype of growing pigs.

The objective of this study was to investigate the secretion of pancreatic enzymes and antibacterial activity in weaned pigs of three pure breeds, Pietrain, Duroc and Polish synthetic line 990, to look for eventual differences related to the genotype. Six male pigs of each breed, about 24 kg mean body weight, were equipped with chronic pancreatic duct catheters and duodenal cannulas to assess pure pancreatic juice, and jugular vein catheters for blood withdrawal. Pancreatic juice was collected before and after the morning feeding. Protein output and enzyme activities revealed two distinct profiles: strong manifestation of the prandial phase in Pietrain and line 990 pigs, and weak manifestation in Duroc. The antibacterial activity did not follow the enzyme kinetics, and it was the strongest in pancreatic juice from Pietrain pigs. Postprandial insulinaemia was reduced in the order of: line 990>Pietrain>Duroc. A slight (not significant) tendency towards a reduction of leptin after feeding in synthetic line 990 corresponded with elevated secretion of pancreatic enzymes and plasma insulin. The presented results suggest that the prandial secretion of pancreatic juice differs according to genotype, and the differences may be in part related to release of insulin.

Animals↗
PubMed2003

Physiological and chemical characteristics of antibacterial activity of pancreatic juice.

Attempts were made to find and characterize an antibacterial activity (ABA) factor in porcine pancreatic juice (PJ). Its isolation requires several steps. Since ABA factor was found to be heat resistant, the first step was heating for 30 min at 65 degrees C. Afterwards column chromatography, ethanol precipitation and polyacrylamide gel electrophoresis were involved. Finally, we obtained a pancreatic juice fraction with antibacterial activity against Escherichia coli strain AB1157. In the presence of this fraction the number of living bacterial cells in overnight culture decreased about 10,000 fold and a spot-test gave clearly positive results. The results of analysis suggest that the antibacterial factor is a polypeptide active in a pH range 8.0-8.5, that migrates in polyacrylamide gel electrophoresis as a band under 14,000 Da. Mass spectroscopy analysis of active fraction showed high concentration of porcine pancreatic spasmolytic polypeptide (PSP). In conclusion, a polypeptide controlling bacterial homeostasis has been found in the porcine pancreatic juice.

Animals↗
PubMed2001

Exposure of Escherichia coli to intestinal myoelectrical activity-related electric field induces resistance against subsequent UV(254 nm) (UVC) irradiation.

Survival of Escherichia coli K-12 AB1157 irradiated with UVC (UV(254 nm)) was enhanced after pre-treatment with a low-tension electric field (EF). The EF used was identical to the electrical field generated by the small intestine (myoelectrical migrating complex--MMC), registered in a healthy calf and transmitted into the memory of an EF generator. The EF emitted by the generator was transmitted via electrodes placed in shaken bacterial cultures. The protective effects of the EF on the E. coli survival after exposure to UV were: (i) observed only for the dnaJ(+)dnaK(+) strain, and not for the DeltadnaJdnaK heat shock mutant; (ii) strictly dependent on the temperature at which the bacteria were grown; (iii) most obvious when the bacteria were incubated at 37 degrees C. Moreover, the MMC-related EF and a higher temperature (40 degrees C) show a similar protective effect against UV-irradiation. The results point to the involvement of the heat shock response in the low-tension EF-induced protection of bacterial cells against UVC-irradiation. Additionally, treatment with the MMC-related EF affects total protein contents and their pattern in E. coli cells. The EF-treatment did not show any influence on the level of the argE3(ochre) --> Arg(+) reversions.

Animals↗
PubMed2001

Contribution of E. coli AlkA, TagA glycosylases and UvrABC-excinuclease in MMS mutagenesis.

MMS, an S(N)2 alkylating agent, is a moderate inducer of SOS mutagenesis and adaptive response. Our previous studies have shown that transient starvation of Escherichia coli AB1157argE3 strain causes a decrease of MMS-induced argE3-->Arg(+) reversions and this decrease is accompanied by the disappearance of the Fpg protein sensitive sites on plasmids isolated from MMS-treated and subsequently starved bacteria. This suggests that in such cells the mutation frequency decline (MFD) repair takes place. Here, we study the relation between MMS-induced mutagenesis as well as mutation frequency decline during starvation, and the repair of alkylated bases and AP-sites by base and nucleotide excision repair systems. In the AB1157alkA(-) strain, MMS-induced mutagenesis was over five-fold higher than in the wild type strain and no MFD repair occurred during starvation. Surprisingly, the lack of TagA glycosylase diminished MMS mutagenesis and accelerated the MFD effect. However, in double tagA(-)alkA(-) mutant, the frequency of Arg(+) reversions increased over 10-fold during 60 min of aminoacid starvation after MMS-treatment. Lack of the uvrA gene function did not affect the MMS-induced mutation rate and MFD in AB1157alkA(+)tagA(+). Starvation of MMS treated AB1157tagAalkAuvrA triple mutant caused a decrease of mutation frequency almost to the level of spontaneous mutation rate. Examination of the repair of 3-MeAde, 7-MeGua and AP sites during starvation using repair glycosylases and plasmids isolated from MMS-treated and starved bacteria revealed that in E. coli uvr(+) but tagAalkA strain, neither 3-MeAde nor 7-MeGua were repaired during 60 min starvation and these persistent lesions could be responsible for the induction of the SOS system and an increase in mutation rate during starvation. In the triple tagAalkAuvrA mutant the repair of 3-MeAde, 7-MeGua and AP sites was carried out effectively and this could explain the observed decrease in the mutation rate during starvation. These results suggest that only in the absence of the "first choice" repair enzymes TagA, AlkA glycosylases and UvrABC excinuclease, a third error-free repair system of alkylated bases is activated. In the absence of only TagA and AlkA glycosylases, UvrABC excinuclease mediates activation of the SOS response, and this results in an increase of mutagenesis induced by the presence of alkylated bases in DNA.

Bacterial Proteins↗
PubMed2001

Influence of intestinal myoelectrical activity on the growth of Escherichia coli.

Intestinal bacteria, particularly those adhering to intestinal epithelial cells, are exposed to electric fields and currents generated by the muscular activity of the small intestine. This activity displays a regular pattern known as the myoelectrical migrating complex (MMC). In order to explore the possibility that these endogenous electric fields could affect bacterial growth, a digitised duodenal signal obtained via serosal electrodes from a healthy calf was recorded and then applied via platinum electrodes to Escherichia coli cultures. The culture tubes were placed within a Faraday shield, incubated at 37 degrees C with shaking, and stimulated by the electric current for 5 or 8 h. The growth of E. coli stimulated by the electric current was significantly altered compared to those of non-stimulated controls: after a period of intensive growth, inhibition of cell division was observed. This was not the case when the bacteria with lon mutation were used. Moreover, synchronic bacterial culture could not be achieved in the presence of the MMC-related electric field. These results suggest that the myoelectrical activity of the duodenum, through action on cell membrane, can affect cell division of intestinal bacteria.

Animals↗
PubMed2000

Fats infused intraduodenally affect the postprandial secretion of the exocrine pancreas and the plasma concentration of cholecystokinin but not of peptide YY in growing pigs.

In pigs, the spontaneous secretion of the exocrine pancreas and the release of cholecystokinin (CCK) and peptide YY (PYY) after intraduodenal infusion of fully saturated synthetic fats differing in chain length was studied. Growing pigs (n = 6) were prepared with pancreatic duct catheters, duodenal T-cannulas and catheters placed in the jugular vein. The pigs were fed 2 g/100 g body twice daily. Beginning with the morning feeding, a medium-chain triglyceride (MCT: glycerol tricaprylate), a long-chain triglyceride (LCT: glycerol tristearate) or saline was infused at a rate of 0.1 g/100 g body. Pancreatic juice was collected, beginning 1 h preprandially until 3 h postprandially. Blood samples were obtained 15 min preprandially and 15, 45, 90 and 150 min postprandially. The infusion of MCT evoked a change in the trend of the curve for the volume of secretion of pancreatic juice, lipase and colipase concentrations and outputs. The trend of the curve did not change over time for CCK and PYY. Differences between the trends of the curves for the saline and MCT treatment were observed for volume of secretion, protein output, lipase content and output, trypsin and colipase output. Differences in the trends of the curves between MCT and LCT were obtained for the outputs of protein, lipase and colipase. Plasma CCK levels were lower as a result of the MCT treatment compared with the saline and LCT treatments. The results suggest an immediate, distinguished response of the porcine exocrine pancreas to fats differing in chain length.

Animals↗
PubMed2000

Reversion of argE3 ochre strain Escherichia coli AB1157 as a tool for studying the stationary-phase (adaptive) mutations.

Adaptive (starvation-associated) mutations occur in non-dividing cells and allow growth under the selective conditions imposed. We developed a new method for the determination of adaptive mutations in Escherichia coli. The system involves reversion to prototrophy of the argE3OC mutation and was tested on AB1157 strains mutated in the mutT and/or mutY genes. The bacteria that mutated adaptively grow into colonies on minimal medium plates devoid of arginine (starvation conditions) when incubated longer than 4 days. Using the replica plating method we solved the problem of discrimination between growth-dependent and adaptive argE3-->Arg+ revertants. Phenotype analysis and susceptibility of the Arg+ revertants to a set of T4 phage mutants create an additional possibility to draw a distinction between these two types of Arg+ revertants.

Adaptation, Physiological↗
PubMed1998

Mutation frequency decline in MMS-treated Escherichia coli K-12 mutS strains.

It has been shown that the decline in mutant frequency (MFD) (argE3(ochre)-->Arg+) which occurs in MMS-treated and then transiently starved AB1157 Escherichia coli K-12 cells concerns revertants which arose by supL suppressor formation in a process which is umuDC dependent. Here we have examined whether MMS-induced Arg+ revertants are susceptible to decline when bacteria are deficient in mismatch repair. We show that there is an absence of MFD in MMS-treated M1 (mutS) and in EC2416 (mutS delta umuDC) cells defective in mismatch repair which is associated with a change in the spectrum of MMS-induced mutations formed. In contrast to AB1157, transformation of M1 (mutS) bacteria with plasmids harbouring various combinations of umuD(D')C genes does not enhance the level of MMS-induced mutations but may influence the proportion of supL mutations. These supL mutations show MFD. Repair processes under MFD conditions were confirmed by analysis of plasmid DNA isolated from MMS-treated bacteria at different stages of their starvation and digestion with Fpg protein.

Adenosine Triphosphatases↗
PubMed1998

The role of mutation frequency decline and SOS repair systems in methyl methanesulfonate mutagenesis.

Methyl methanesulfonate (MMS) is an SN2 type alkylating agent which predominantly methylates nitrogen atoms in purines. Among the methylated bases 3meA and 3meG are highly mutagenic and toxic. The excision of these lesions leads to the formation of apurinic (AP) sites and subsequently to AT-->TA or GC-->TA transversions. The in vivo method based on phenotypic analysis of Arg+ revertants of Escherichia coli K12 and sensitivity to T4 nonsense mutants has been used to estimate the specificity of MMS induced mutations. In the E. coli arg-his-thr- (AB1157) strain MMS induces argE3(oc)-->Arg+ revertants of which 70-80% arise by supL suppressor formation as a result of AT-->TA transversions. The remaining 20-30% arise by supB and supE(oc) suppressor formation as a result of GC-->AT transitions. The level of AT-->TA transversions decreases during starvation. This is a consequence of action of the repair mechanism called mutation frequency decline. This system which is a transcription coupled variant of nucleotide excision repair was discovered in UV induced mutations. We describe the mutation frequency decline phenomenon for MMS mutagenesis. MMS is a very efficient inducer of the SOS response and a umuDC dependent mutagen. In MMS treated E. coli cells mutated in umuDC genes the class of AT-->TA transversions dramatically diminishes. A plasmid bearing UmuD(D')C proteins can supplement chromosomal deletion of umuDC operon: a plasmid harbouring umuD'C is more efficient in comparison to that harbouring umuDC. Moreover, plasmids isolated from MMS treated and transiently starved E. coli AB1157 cells harbouring umuD(D')C genes have shown the repair of AP sites by a system which involves the UmuD'C or at least UmuD' protein.

Animals↗
PubMed1996

MMS-induced mutagenesis and DNA repair in Escherichia coli dnaQ49: contribution of UmuD' to DNA repair.

dnaQ-encoded epsilon subunit of DNA polymerase III, possesses 3',5' exonuclease (proofreading) activity, and is a fidelity factor of polymerase III holoenzyme. It is assumed that during SOS-induced mutagenesis, UmuD', UmuC, and RecA may suppress DnaQ proofreading activity, and allow for translesional DNA synthesis at the cost of fidelity of replication. In this report SOS-dependent, MMS-induced mutagenesis and DNA repair were tested in E. coli dnaQ49 strains. Bacteria were transformed with various pDNAs harboring compilation of the umuD(D')C genes, and the influence of plasmids on mutagenesis (argE3-->Arg+) and DNA repair was tested. DNA damage and repair were tested in plasmid DNA grown in MMS-treated bacteria and isolated either immediately after MMS treatment, or after starving the cells (MFD conditions) for 30 and 60 min, then nicking activity of Fpg protein on plasmid DNAs was analyzed. It has been found that (i) repair of MMS-induced lesions depends on umuD'C, umuD' (and to much less degree, on umuDC) genes encoded in pDNA; (ii) MMS-induced mutations, in contrast to DNA repair, are highest in the cells transformed with pDNA harboring umuDC, and lowest or zero in cells with plasmids harboring umuD'C. It is postulated that UmuD'C or UmuD' proteins play a role in the repair of damaged DNA and/or in maintenance of DNA integrity. The kinetics of these processes (perhaps due to introducing too many of the lesions) seems to be different in E. coli dnaQ+ and dnaQ cells, and probably this is a reason that (iii) MMS-induced mutations in dnaQ49 strains are not subject to MFD.

Arginine↗
PubMed1996

Conformation of plasmid DNA from Escherichia coli deficient in the repair systems protecting DNA from 8-oxyguanine lesions.

8-Oxyguanine (8ohG) is a major oxidation product of guanine and a biomarker of oxidative stress in mammal. We have attempted to estimate the level of 8ohG residues in plasmid DNA (pGW2123 and pBR322) grown in various bacterial strains (fpg, mutY, or mutT, plus mutT fpg and mutT mutY double mutants) differing in the system protecting cells against the mutagenic effects of 8ohG in DNA. The method was based on digestion of plasmid DNA with Fpg, and agarose gel electrophoresis. Fpg converts pDNA from covalently closed circular to the open circular (ccc-->oc) form of pDNA when there is at least one 8ohG, or apurinic site, per ccc pDNA molecule. It was found that: i) the content of 8ohG in pDNA grown in any of the tested bacteria is below one 8ohG per 10(4) base pairs; ii) a substantial part of pGW2123 is isolated from the bacteria in the oc form; iii) the ratio of oc/ccc in pGW2123 depends on the bacterial host and is the lowest when the plasmid was harvested from mutY- deficient cells; iv) pBR322, unlike pGW2123, is isolated predominantly in the ccc form; and v) of the pBR322 grown in the tested bacteria apparently the most resistant to Fpg digestion was pBR322 grown in the mutY strain. It is proposed that this reflects the compact structure of pDNAs when they are grown in bacteria deficient in mutY gene product.

DNA Repair↗
PubMed1994

The frequency of MMS-induced, umuDC-dependent, mutations declines during starvation in Escherichia coli.

It has been found that the level of methyl methanesulfonate (MMS)-induced mutation in Escherichia coli is dependent on the level of UmuD(D')C proteins. The frequency of argE(ochre)-->Arg+ mutations (which occur predominantly by AT-->TA transversions) and RifS-->RifR mutations is much higher when UmuDC or UmuD'C are overproduced in the cell. When MMS-treated bacteria were starved for progressively longer times and hence the expression of mutations delayed, the level of mutations observed progressively declined. This same treatment had no effect on the degree of SOS induction. Examination of plasmid DNAs, isolated from MMS-treated cells, for their sensitivity to the specific endonucleases Fpg and Nth revealed that MMS causes formation of abasic sites, which are repaired during cell starvation. It is assumed that, in non-dividing cells, apurinic sites are mostly repaired by RecA-mediated recombinational repair. This pathway, which is error-free, is compared with the processing pathway in metabolically active cells, where translesion synthesis by the UmuD'2C-RecA-DNA polymerase III holoenzyme complex occurs; this latter pathway is error-prone.

Apurinic Acid↗
PubMed1993

Some aspects of EMS-induced mutagenesis in Escherichia coli.

AB2497 and its mutS and umuDC derivatives were EMS-treated at the stationary phase and specificity of mutation measured. It was found that: (i) in mutS+ cells EMS induces predominantly GC-->AT transitions (by supB or supE(oc) formation) and in mutS- cells mainly AT-->TA transversions (by supL(NG) formation); (ii) transversions of AT-->TA are umuDC-dependent and mutational specificity is biased towards AT-->GC transitions in mutS- umuDC- strains. When mutS- umuDC- cells were transfected with plasmids bearing umuD'C or umuDC genes, mutational specificity was again biased towards AT-->TA transversions; (iii) experiments with bacteria bearing umuC::lacZ or recA::lacZ fusions suggest that processing of UmuD-->UmuD' might be poorer in EMS-treated mutS- than in mutS+ cells.

Bacterial Proteins↗
PubMed1992

Mutagenesis of Escherichia coli: a method for determining mutagenic specificity by analysis of tRNA suppressors.

A method for estimating mutagenic specificity in Escherichia coli (argE3, hisG4, thr-1, supE44), based upon the isolation of Arg+ or His+ revertants and identification of tRNA suppressors, is described. The method gives an insight not only into mutagenic pathways but also into the functioning of tRNA. With N-methyl-N'-nitro-N-nitrosoguanidine, 98% of mutations are GC----AT transitions. With N4-hydroxycytidine, 100% are AT----GC transitions. With hydroxylamine, apart from GC----AT transitions, approximately 30% of Arg+ revertants are formed by GC (or AT)----TA transversions. When the chemistry of the mutagenic attack is known, the method allows us to discriminate whether mutations occur on the transcribed or non-transcribed strands of DNA. It has been found that reversion of argE3 to Arg+ is a better monitor of mutagenic pathways than reversion of hisG4 to His+.

Arginine↗