Alterations in the liver of intrauterine growth retarded piglets may predispose to development of insulin resistance and obesity in later life.

Intrauterine growth retardation (IUGR) leads to increased predisposition to metabolic syndrome in adult life but the mechanisms remain obscure. Considering a significant number of functional similarities, IUGR piglets appear to be a good model to study the development of this syndrome in humans. The aim of the present study was to investigate the ultrastructure and proteomic profile of the liver in IUGR pig neonates to discover early markers of predisposition to obesity and insulin resistance. In our study intestine and liver tissue samples were investigated in 7 day old IUGR and normal body weight (NBW) littermate piglets using histometry, mass spectrometry, in-tissue cytometry analysis and confocal microscopy. Compared to NBW, the liver in IUGR neonates was characterized by a significantly enhanced ratio of Kupffer cells to hepatocytes and insulin receptor abundance as well as higher percentages of cells expressing receptors for adipokines (resistin and adiponectin), increased expression of TNF-α (as marker of inflammation), and increased expression of insulin receptor and uncoupling protein 3 (UCP3). Moreover, NBW and IUGR differed in proteomic profile, including protein metabolism (proteasomes, cathepsin D, phermitin, phosphoglucomutase), carbohydrate metabolism (hexokinase 1, phosphoglucokinase, galactokinase, aldolase B, glucose-6-phosphate isomerase), oxidative stress and chromatin organization and DNA uptake (histones, lamin a/c). Reduction of hepatocyte numbers concomitant with significant modifications of expression of key hormones and enzymes for protein and carbohydrate metabolism in IUGR neonates may predispose to insulin resistance and obesity in adult life.

Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes.

When DNA molecules are injected into Xenopus oocyte nuclei, they can recombine with each other. With bacteriophage lambda DNAs, it was shown that this recombination is stimulated greatly by introduction of double-strand breaks into the substrates and is dependent on homologous overlaps in the recombination interval. With plasmid DNAs it was shown that little or no recombination occurs between circular molecules but both intra- and intermolecular events take place very efficiently with linear molecules. As with the lambda substrates, homology was required to support recombination; no simple joining of ends was observed. Blockage of DNA ends with nonhomologous sequences interfered with recombination, indicating that ends are used directly to initiate homologous interactions. These observations are combined to evaluate possible models of recombination in the oocytes. Because each oocyte is capable of recombining nanogram quantities of linear DNA, this system offers exceptional opportunities for detailed molecular analysis of the recombination process in a higher organism.

Heat shock response in gastrointestinal tract.

Heat shock response is one of the defense mechanisms common to eukaryotic and prokaryotic cells. The highly conserved and ubiquitous heat shock proteins (HSPs) are essential for cell survival during stress. Stress tolerance, i.e., adaptation of cells to stress conditions, is a characteristic feature of heat shock response. The lumen of the gastrointestinal tract is an external environment to the body. Epithelium of the digestive tract is exposed to various stress factors inducing the heat shock response, e.g., bacteria and their toxins, food borne chemical compounds, drugs and diet deficiencies. Other factors like plant lecitins, glutamine or short fatty acids are mild stressors and can modulate the heat shock response in cells. All these factors are presumed to influence the normal microflora that is an integral part of the digestive tract. This review is focused on the induction/modulation of heat shock proteins expression in the epithelium of the gastrointestinal tract by various factors, on the protective role of HSPs and mechanisms leading to stress protection inside the gut. Heat shock response is one of the key mechanisms of maintenance of gastrointestinal tract homeostasis. It is involved in pathogenic bacteria adaptation to life in the digestive tract, especially in colony formation and in their role in infectious processes.

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.

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.

Evaluation of the Escherichia coli HK82 and BS87 strains as tools for AlkB studies.

Within a decade the family of AlkB dioxygenases has been extensively studied as a one-protein DNA/RNA repair system in Escherichia coli but also as a group of proteins of much wider functions in eukaryotes. Two strains, HK82 and BS87, are the most commonly used E. coli strains for the alkB gene mutations. The aim of this study was to assess the usefulness of these alkB mutants in different aspects of research on AlkB dioxygenases that function not only in alkylated DNA repair but also in other metabolic processes in cells. Using of HK82 and BS87 strains, we found the following differences among these alkB(-) derivatives: (i) HK82 has shown more than 10-fold higher MMS-induced mutagenesis in comparison to BS87; (ii) different specificity of Arg(+) revertants; (iii) increased induction of SOS and Ada responses in HK82; (iv) the genome of HK82, in comparison to AB1157 and BS87, contains additional mutations: nalA, sbcC, and nuoC. We hypothesize that in HK82 these mutations, together with the non-functional AlkB protein, may result in much higher contents of ssDNA, thus higher in comparison to BS87 MMS-induced mutagenesis. In the light of our findings, we strongly recommend using BS87 strain in AlkB research as HK82, bearing several additional mutations in its genome, is not an exact derivative of the AB1157 strain, and shows additional features that may disturb proper interpretation of obtained results.

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.

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.

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.

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.

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.

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.

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.

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.

Intestinal MMC-related electric fields and pancreatic juice control the adhesion of Gram-positive and Gram-negative bacteria to the gut epithelium--in vitro study.

The adhesion of six different Lactobacillus and Lactococcus and three pathogenic Escherichia and Salmonella strains was studied using Caco-2 cell line. In this in vitro model system the influence of weak electric field (EF) on bacterial adhesion was tested. The EF source was the in vitro reconstruction of spiking potentials recorded in the duodenum of a healthy calf during one myoelectrical migration complex (MMC) cycle. The ability to adhere to Caco-2 cells of bacteria belonging to two groups, Gram-positive lactobacilli and lactococci, and Gram-negative Escherichia and Salmonella differed considerably. The pathogenic bacteria adhered better to well-differentiated Caco-2 cells whereas lactobacilli and lactococci displayed better adhesion to non-differentiated Caco-2 cells. In the presence of MMC-related EF an increased adhesion of Lactobacillus and Lactococcus but not of Salmonella enterica s. Enteritidis and E. coli 269 to Caco-2 cells was observed. Two later strains adhered even less in the presence of EF. The same tendency was found in the presence of pancreatic juice in a cell medium. In conclusion, the myoelectric component of the small intestinal motility, the MMC-related EF, and pancreatic juice may increase the ability of lactic acid bacteria to adhere to GI epithelial cells, creating better environmental conditions for colonization of the intestine and competition with Gram-negative pathogens.

Transcription of bacteriophage PM2.

Transcription of bacteriophage PM2 after infection of Alteromonas espejiana BAL-31 was examined. A wave of PM2 late transcription was observed 22 to 44 min after infection. This transcription was chloramphenicol- and rifampicin-sensitive. Regions of PM2 DNA transcribed with high efficiency were determined by DNA--RNA hybridization and Southern's technique.

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.