Roles of ExoI and SbcCD Nucleases in “Reckless” DNA Degradation in recA Mutants of Escherichia coli

Exponentially growing recA mutant cells of Escherichia coli display pronounced DNA degradation that starts at the sites of DNA damage and depends on RecBCD nuclease (ExoV) activity. As a consequence of this “reckless” DNA degradation, populations of recA mutants contain a large proportion of anucleate cells. We have found that both DNA degradation and anucleate-cell production are efficiently suppressed by mutations in the xonA (sbcB) and sbcD genes. The suppressive effects of these mutations were observed in normally grown, as well as in UV-irradiated, recA cells. The products of the xonA and sbcD genes are known to code for the ExoI and SbcCD nucleases, respectively. Since both xonA and sbcD mutations are required for strong suppression of DNA degradation while individual mutations have only a weak suppressive effect, we infer that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells. We suggest that their roles might be in processing (blunting) DNA ends, thereby producing suitable substrates for RecBCD binding.The RecA protein plays a central role in homologous recombination and recombinational DNA repair in Escherichia coli, as well as in other bacterial species. It catalyzes the key stages of the recombination process—homologous pairing and DNA strand exchange. Cells carrying null mutations in the recA gene are completely deficient for homologous recombination and are extremely sensitive to DNA-damaging agents (for a review, see references 21, 24, and 25). Populations of recA null mutants contain a large proportion (50 to 60%) of nonviable cells, reflecting the inability of these mutants to repair spontaneously occurring DNA damage (31). Also, exponentially growing recA cells display pronounced spontaneous DNA degradation that presumably starts at the sites of DNA damage and that depends on RecBCD nuclease (ExoV) activity (5, 48). This phenotype of recA cells is aggravated after DNA-damaging treatment, such as UV irradiation (48).According to the present data, the majority of RecA-catalyzed DNA transactions in E. coli start with binding of the RecA protein onto single-stranded DNA (ssDNA) substrates. This binding is mediated by the RecBCD and/or RecFOR protein, which helps RecA to overcome hindrance imposed by the SSB protein during competition for the DNA substrate. The RecBCD and RecFOR proteins begin RecA polymerization on ssDNA, giving rise to a nucleoprotein filament that is indispensable for further recombination reactions (3, 33; reviewed in reference 44).The RecBCD enzyme is crucial for initiation of recombinational processes at double-stranded DNA (dsDNA) ends (or breaks [DSBs]) in wild-type E. coli (a set of reactions known as the RecBCD pathway) (9, 43, 44). Upon recognizing a blunt or nearly blunt dsDNA end and binding to it, RecBCD acts as a combination of powerful helicase and nuclease, thus unwinding and simultaneously degrading both strands of the DNA duplex. After encountering a specific octanucleotide sequence designated Chi, the strong 3′-5′ nuclease activity of the enzyme is attenuated and a weaker 5′-3′ nuclease activity is upregulated (1). This Chi-dependent modification allows RecBCD to create a long 3′ ssDNA tail and to direct the loading of RecA protein onto it (2, 3). In vivo data suggest that this transition of RecBCD from a nuclease to a recombinase mode of action requires the presence of the RecA protein, suggesting that the two proteins might interact (27).In wild-type E. coli cells, the RecFOR protein complex works predominantly on DNA gaps, which may arise in chromosomes due to replication forks passing over the noncoding lesions (e.g., UV-induced pyrimidine dimers) or may be present in replication forks stalled at different obstacles in DNA (44). On the other hand, the RecFOR complex has an important role in recBC sbcBC(D) mutant cells, replacing the RecA-loading activity of RecBCD during recombination reactions starting from dsDNA ends. Recombination reactions mediated by RecFOR proteins are termed the RecF (or RecFOR) pathway (44).Cells mutated in the recB and/or recC gene exhibit strong deficiency in conjugational and transductional recombination, as well as in the repair of DSBs (8, 21). These defects can be rectified by extragenic sbcB and sbcC(D) suppressor mutations that inactivate two nucleases, thus enabling full efficiency of the RecF pathway on dsDNA ends (21, 44). The sbcB gene (also designated xonA) encodes exonuclease I (ExoI), the enzyme that digests ssDNA in the 3′-5′ direction (23). The sbcC and sbcD genes encode subunits of the SbcCD nuclease, which acts both as an endonuclease that cleaves hairpin structures and as an exonuclease that degrades linear dsDNA molecules (10, 11). Inactivation of either of the two subunits leads to the loss of SbcCD enzyme activity (18).The exact mechanism of activation of the RecF pathway by sbc mutations is not completely understood. A plausible explanation is that inactivation of ExoI and SbcCD nucleases is necessary to prevent the degradation of recombinogenic 3′ DNA ends created in a RecBCD-independent manner (8, 23, 38, 45, 46). It was recently shown that the sbcB15 mutant allele (encoding a protein without nucleolytic activity) (37) is a better suppressor of the RecBCD⁻ phenotype than an sbcB deletion (50), suggesting that some nonnucleolytic activity of ExoI may also contribute to the efficiency of the RecF pathway (46, 50).ExoI and SbcCD are usually viewed as enzymes with inhibitory roles in recombination due to their deleterious actions on the RecF pathway. However, some results suggest that these enzymes could also have stimulatory roles in recombination reactions proceeding on the RecBCD pathway. Genetic experiments with UV-irradiated E. coli cells indicated that ExoI and SbcCD might be involved in blunting radiation-induced DNA ends prior to RecBC(D) action (38, 45, 46). Such a role of ExoI and SbcCD seems to be particularly critical in recD recF mutants, in which the majority of DSB repair depends on the RecBC enzyme (38). It was also suggested that the blunting roles of the two nucleases may be required during conjugational recombination (16, 46).In this work, we studied the effects of sbcB (xonA) and sbcD mutations on DNA degradation occurring spontaneously in exponentially growing recA mutant cells, as well as on DNA degradation induced in recA mutants by UV irradiation. We have demonstrated that in both cases DNA degradation is strongly reduced in recA mutants that carry in addition a combination of xonA and sbcD null mutations. The results described in this paper suggest that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells.     

Foliar and root treatments of cucumber with potassium naphthenate: Antioxidative responses

This work presents a study of the effect of foliar and root application of low concentrations (0.1?C10 ??M) of potassium naphthenate on the antioxidative status of cucumber (Cucumis sativus L.), assessed for both local and systemic organs. Changes in the contents of proline and glutathione indicate that the treatment of plants with potassium naphthenate can be characterized as a mild abiotic stress. The antioxidative system of cucumber plants is sensitive to such treatment, since organs directly exposed to the chemical showed a decrease in total antioxidant activities and an increase in peroxidation. In the organs that were not directly treated, an increase in the total antioxidative activity was observed only at the lowest naphthenate concentration while at higher concentrations this activity tended to decrease. As far as the activities of antioxidant enzymes (guaiacol peroxidase, superoxide dismutase, catalase) are concerned, the responses observed differed between enzymes for a given treatment, but showed similar trends within treated local and untreated systemic organs.     

Multiple-copy cluster-type organization and evolution of genes encoding O-methyltransferases in the apple

Plant O-methyltransferases (OMTs) play important roles in secondary metabolism. Two clusters of genes coding for caffeic acid OMT (COMT) have been identified in the apple genome. Three genes from one cluster and two genes from another cluster were isolated. These five genes encoding COMT, designated Mdomt1-Mdomt5 (GenBank accession nos. DQ886018-DQ886022), were distinguished by a (CT)(n) microsatellite in the 5'-UTR and two transposon-like sequences present in the promoter region and intron 1, respectively. The transposon-like sequence in intron 1 unambiguously traced the five Mdomt genes in the apple to a common ancestor. The ancestor must have undergone an initial duplication generating two progenitors, and this was followed by further duplication of these progenitors resulting in the two clusters identified in this study. The distal regions of the transposon-like sequences in promoter regions of Mdomt genes are capable of forming palindromic hairpin-like structures. The hairpin formation is likely responsible for nucleotide sequence differences observed in the promoter regions of these genes as it plays a destabilizing role in eukaryotic chromosomes. In addition, the possible mechanism of amplification of Mdomt genes in the apple genome is also discussed.     

Quantification of climate tourism potential of Croatia based on measured data and regional modeling

Tourism is one of the most important economic sectors in Croatia. The Adriatic coast is a popular travel destination for tourists, especially during the summer months. During their activities, tourists are affected by atmospheric conditions and therefore by weather and climate. Therefore, it is important to have reliable information about thermal conditions as well as their impacts on human beings. Here, the climate tourism potential of Croatia is presented and quantified on the basis of three selected stations in different climatic regions. The physiologically equivalent temperature is used for analysis as well as other climatic parameters relevant for tourism and recreation. The results already point to hot conditions for outdoor activities in summer during afternoons, especially along the coast but also for continental regions, resulting in a reduction of the climate tourism potential. In the future, this trend looks set to increase, possibly leading to a changing tourism sector in Croatia requiring adaptation and new strategies.     

Atomic Force Microscopy Stiffness Tomography on Living Arabidopsis thaliana Cells Reveals the Mechanical Properties of Surface and Deep Cell-Wall Layers during Growth

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content.     

Differential metabolic consequences of fumarate hydratase and respiratory chain defects

Defects of the oxidative ATP production pathway lead to an amazing variety of disease phenotypes, ranging from childhood encephalomyopathies to hereditary tumor formation. A key enzyme of tricarboxylic cycle, fumarate hydratase (FH), is involved in encephalopathies, but also in leiomyoma formation, and occasionally also in various types of cancer. MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and NARP (neuropathy ataxia retinitis pigmentosa) are progressive neurological disorders, caused by mitochondrial DNA mutations and respiratory chain (RC) deficiency. These diseases lead to disability and premature death, but not to tumorigenesis. We studied the cellular consequences of FH and RC deficiencies, aiming to identify general responses to energy metabolism defect and those specific for FH-deficiency, suggestively connected to tumorigenesis. Unlike in RC deficiency, the FH-deficient diploid human fibroblasts showed no signs of oxidative stress, but had a reduced redox state with high glutathione levels. The cytoplasmic FH isoform, previously described, but with an unknown function, was completely lacking in all FH-deficient lines. Fumarate was increased in two of our FH-lines, but accumulation of HIF-1alpha was not detected. Glycolysis was induced in both MELAS and in FH-deficiency. Accumulation of fumarate in primary fibroblasts did not activate a hypoxia response, suggesting that hypoxia activation due to fumarate accumulation may be a tissue-specific response. The lack of cytoplasmic form of FH and the reduced redox environment were typical for all FH-mutant lines, and their role in FH-related tumorigenesis requires further attention.     

Genetically different wine yeasts isolated from Austrian vine-growing regions influence wine aroma differently and contain putative hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii

To evaluate the influence of the genomic properties of yeasts on the formation of wine flavour, genotypic diversity among natural Saccharomyces cerevisiae strains originating from grapes collected in four localities of three Austrian vine-growing areas (Thermenregion: locations Perchtoldsdorf and Pfaffst?tten, Neusiedlersee-Hügelland: location Eisenstadt, Neusiedlersee: location Halbturn) was investigated and the aroma compounds produced during fermentation of the grape must of 'Grüner Veltliner' were identified. Amplified fragment length polymorphism analysis (AFLP) showed that the yeast strains cluster in four groups corresponding to their geographical origin. The genotypic analysis and sequencing of the D1/D2 domain of 26S rRNA encoding gene and ITS1/ITS2 regions indicated that the Perchtoldsdorf strains were putative interspecies hybrids between S. cerevisiae and Saccharomyces kudriavzevii. Analysis of the aroma compounds by GS/MS indicated a region-specific influence of the yeasts on the chemical composition of the wines. The aroma compound profiles generated by the Perchtoldsdorf strains were more related to those produced by the Pfaffst?tten strains than by the Eisenstadt and Halbturn strains. Similar to the Pfaffst?tten yeasts, the putative hybrid strains were good ester producers, suggesting that they may influence the wine quality favourably.     

The prevalence of cardiovascular disease risk factors in patients from Croatian Zagorje County treated at Department of Medicine, Zabok General Hospital from 2000 to 2006

The aim of the study was to assess the prevalence of risk factors for cardiovascular disease in patients treated for coronary heart disease (CHD) at Department of Medicine, Zabok General Hospital during the 2000-2006 period. Cardiovascular diseases are a group of diseases that occur due to arterial. The risk factors that lead to the development and occurrence of cardiovascular disease are hypertension, cigarette smoking, hyperholesterolemia, hypertriglyceridemia, diabetes mellitus and positive family history. Additional factors favoring the occurrence of cardiovascular disease include overweight, inadequate physical activity, and emotional stress. Data on all patients hospitalized and diagnosed with CHD at Department of Medicine, Zabok General Hospital during the 2000-2006 period were analyzed for the prevalence of risk factors for CHD, i.e. hypertension, cigarette smoking, hypercholesterolemia, hypertriglyceridemia, diabetes mellitus and positive family history of cardiovascular disease. Hypercholesterolemia was defined by a cholesterol level higher than 5.1 mmol/L, hypertension from history data and blood pressure measurement on admission greater than 140/90 mmHg, diabetes mellitus from history data, and hypertriglyceridemia by a triglyceride level greater than 1.7 mmol/L. Information on heredity and cigarette smoking was collected from history and a questionnaire filled out on admission. All laboratory values were determined on patient admission to the hospital. Analysis of the risk factors for CHD recorded in patients from Zagorje County during the 2000-2006 period revealed hypertension to be the most common risk factor in our patients. According to sex, CHD was found to show a male preponderance. According to age at admission, CHD predominated in the > 70 age group, which accounted for one third of all patients, followed by a comparable proportion of the 50-60 and 60-70 age groups, i.e. still active population groups. As CHD is one of the leading health threats worldwide, estimated to remain so at least by 2020, it is fully justified to invest all efforts in the study of cardiovascular disease. New research projects should be focused on the prevention and early detection of the disease, improvement of diagnosis procedures, introduction of novel therapeutic options, use of new concepts, and due survey of the measures taken. CHD poses great socioeconomic burden upon every community in industrialized societies because of the ever younger age at onset. Actions should be taken to improve awareness of the CHD risks and morbidity in the population at large, stimulating favorable lifestyle and dietary modifications, and one's own health awareness, in order to upgrade the control of risk factors for and morbidity of cardiovascular disease.     

Estimation of phylogenetic relationships within the Ascomycota on the basis of 18S rDNA sequences and chemotaxonomy

Small subunit rRNA gene sequences (18S rDNA), cell wall carbohydrate composition and ubiquinone components were analysed within a larger number of ascomycetous yeasts and dimorphic fungi to validate their congruence in predicting phylogenetic relationships. The glucose-mannose pattern distinguishes the Hemiascomycetes from the Euascomycetes and the Protomycetes which are characterised with the glucose-mannose-galactose-rhamnose-(fucose) profile. The glucose-mannose-galactose pattern was found in the cell walls of all the three classes. Different coenzyme Q component (CoQ5 to CoQ10) were found within the representatives of the Hemiascomycetes. Whereas CoQ9, CoQ10 and CoQ10H2 predominate within the Euascomycetes, CoQ9 and CoQ10 characterise the Protomycetes. Chemotaxonomic studies coupled with additional molecular and co-evolution studies support the idea that the Hemiascomycetes occupy a basal position in the phylogeny of Ascomycota. These results are not in line with the phylogenetic studies based on the sequences of 18S rRNA encoding gene. The maximum parsimony analysis indicated that Hemiascomycetes and Protomycetes might represent sister groups, opposing to the earlier reported results, where the Archiascomycetes (Protomycetes) or the Hemiascomycetes had been considered to be the most primitive ascomycetous fungi. Instead of the class Archiascomycetes, the term Protomycetes was introduced reflecting much better the properties of the whole class.     

Effect of acute heat stress on rat adrenal cortex — a morphological and ultrastructural study

The stereological structure of rat adrenal gland was analysed by light and electron microscopy after an acute (60 min) exposure to high ambient temperature (38°C). Under these conditions a significant increase in plasma corticotrophin (ACTH), serum corticosterone and aldosterone levels were observed. Histological and stereological investigation at light microscopy showed significant decrease in volume density of capsule and zona glomerulosa, increase in volume of fasciculata cells, and decrease of numerical density of zona fasciculata cells and mean diameter of blood vessels. At the ultrastructural level, volume density of nuclei and mitochondria of zona glomerulosa cells were significantly increased and that of lipid droplets decreased. Volume density of mitochondria of fasciculata cells was significantly increased, while number of lipid droplets per μm² of cell was reduced. In the cells of zona reticularis significant increase in the number of lipid droplets was found. The response of zona glomerulosa may be interpreted as immediate reaction to dehydration, while alterations detected in zona fasciculata, which were less extensive, were related to purely stressogenic effects of high ambiental temperature.