APE1 overexpression in XRCC1-deficient cells complements the defective repair of oxidative single strand breaks but increases genomic instability

XRCC1 protein is essential for mammalian viability and is required for the efficient repair of single strand breaks (SSBs) and damaged bases in DNA. XRCC1-deficient cells are genetically unstable and sensitive to DNA damaging agents. XRCC1 has no known enzymatic activity and is thought to act as a scaffold protein for both SSB and base excision repair activities. To further define the defects leading to genetic instability in XRCC1-deficient cells, we overexpressed the AP endonuclease APE1, shown previously to interact with and be stimulated by XRCC1. Here, we report that the overexpression of APE1 can compensate for the impaired capability of XRCC1-deficient cells to repair SSBs induced by oxidative DNA damage, both in vivo and in whole-cell extracts. We show that, for this kind of damage, the repair of blocked DNA ends is rate limiting and can be performed by APE1. Conversely, APE1 overproduction resulted in a 3-fold increase in the sensitivity of XRCC1-deficient cells to an alkylating agent, most probably due to the accumulation of SSBs. Finally, the overproduction of APE1 results in increases of 40% in the frequency of micronuclei and 33% in sister chromatid exchanges of XRCC1⁻ cells. These data suggest that the spontaneous generation of AP sites could be at the origin of the SSBs responsible for the spontaneous genetic instability characteristic of XRCC1-deficient cells.     

Genetic variability in a genomic region with long-range linkage disequilibrium reveals traces of a bottleneck in the history of the European population

The inference of the demographic history of populations from genetic variability data is not only of academic interest. It also provides background information for the identification of genes which may have played a role in human evolution or in the aetiology of human disease. To obtain a clear picture of this background, it is necessary to compare data obtained from a number of genomic loci. Due to its very low recombination rate, the NF1 gene region can be regarded as a further suitable locus. A combined resequencing and SNP typing project in a European population disclosed the presence of only two well separated subgroups of NF1 sequences. Statistical analysis revealed a bimodal distribution of the pairwise differences, a positive value of Tajima’s D and a TMRCA of 700,000 years for the whole sample, and pairwise differences indicative for a growing population and TMRCAs of 130,000 to 150,000 years for the subgroups. Together, the data lead to a model that the recent European population went through a bottleneck during the last 150,000 years of its history. Regarding the given timeframe, this bottleneck could either reflect a speciation event which led to the anatomically modern human (AMH), or a severe reduction of the population size during the emigration of AMHs out of Africa or the immigration into Europe.     

Effect of histamine and cimetidine on retinal and choroidal blood flow in humans

Intravenous administration of histamine causes an increase in choroidal blood flow and retinal vessel diameter in healthy subjects. The mechanism underlying this effect remains to be elucidated. In the present study, we hypothesized that H2 receptor blockade alters hemodynamic effects of histamine in the choroid and retina. Eighteen healthy male nonsmoking volunteers were included in this randomized, double-masked, placebo-controlled two-way crossover study. Histamine (0.32 microg.kg(-1).min(-1) over 30 min) was infused intravenously in the absence (NaCl as placebo) or presence of the H2 blocker cimetidine (2.3 mg/min over 50 min). Ocular hemodynamic parameters, blood pressure, and intraocular pressure were measured before drug administration, after infusion of cimetidine or placebo, and after coinfusion of histamine. Subfoveal choroidal blood flow and fundus pulsation amplitude were measured with laser-Doppler flowmetry and laser interferometry, respectively. Retinal arterial and venous diameters were measured with a retinal vessel analyzer. Retinal blood velocity was assessed with bidirectional laser-Doppler velocimetry. Histamine increased subfoveal choroidal blood flow (+14 +/- 15%, P < 0.001), fundus pulsation amplitude (+11 +/- 5%, P < 0.001), retinal venous diameter (+3.0 +/- 3.6%, P = 0.002), and retinal arterial diameter (+2.8 +/- 4.2%, P < 0.01) but did not change retinal blood velocity. The H2 antagonist cimetidine had no significant effect on ocular hemodynamic parameters. In addition, cimetidine did not modify effects of histamine on choroidal blood flow, fundus pulsation amplitude, retinal venous diameter, and retinal arterial diameter compared with placebo. The present data confirm that histamine increases choroidal blood flow and retinal vessel diameters in healthy subjects. This ocular vasodilator effect of histamine is, however, not altered by administration of an H2 blocker. Whether the increase in blood flow is mediated via H1 receptors or other hitherto unidentified mechanisms remains to be elucidated.     

Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice

Phospholemman (FXYD1), a 72-amino acid transmembrane protein abundantly expressed in the heart and skeletal muscle, is a major substrate for phosphorylation in the cardiomyocyte sarcolemma. Biochemical, cellular, and electrophysiological studies have suggested a number of possible roles for this protein, including ion channel modulator, taurine-release channel, Na(+)/Ca(2+) exchanger modulator, and Na-K-ATPase-associated subunit. We have generated a phospholemman-deficient mouse. The adult null mice exhibited increased cardiac mass, larger cardiomyocytes, and ejection fractions that were 9% higher by magnetic resonance imaging compared with wild-type animals. Notably, this occurred in the absence of hypertension. Total Na-K-ATPase activity was 50% lower in the phospholemman-deficient hearts. Expression (per unit of membrane protein) of total Na-K-ATPase was only slightly diminished, but expression of the minor alpha(2)-isoform, which has been specifically implicated in the control of contractility, was reduced by 60%. The absence of phospholemman thus results in a complex response, including a surprisingly large reduction in intrinsic Na-K-ATPase activity, changes in Na-K-ATPase isoform expression, increase in ejection fraction, and increase in cardiac mass. We hypothesize that a primary effect of phospholemman is to modulate the Na-K-ATPase and that its reduced activity initiates compensatory responses.     

MRL/lpr mice have alterations in brain metabolism as shown with [1H-13C] NMR spectroscopy

Cerebral glucose metabolism and cerebral blood flow are altered in patients with lupus who have neuropsychiatric manifestations. However, the dynamics of changes in glucose metabolism remain unclear. The present study was undertaken using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy to determine the rates of incorporation of glucose into amino acids and lactate via cell-specific pathways in mice with lupus. In the well-established MRL/lpr lupus mouse model, 24-week-old mice had a significant increase of 30-80% (P<0.001) in total brain glutamine, glutamate and lactate concentrations, while alanine, aspartate, N-acetyl aspartate (NAA) and gamma-aminobutyric acid (GABA) remained unchanged as compared to the congenic MRL+/+control mice. Although succinate concentration was increased in lupus brain, it did not reach statistical significance. Furthermore, 13C isotopomer analysis showed a selective increase of de novo synthesis of lactate from [1-(13)C] glucose through glycolysis resulting in 1.5-fold increased fractional 13C enrichments in lactate in MRL/lpr mice. [4-(13)C] Glutamate, which is synthesized mainly by the neuronal pyruvate dehydogenase, was selectively increased, while [2-(13)C] and [3-(13)C] GABA synthesis were decreased by 25% compared to controls. In accordance with the total concentrations, aspartate synthesis remained unaltered in brains of lupus mice, while alanine synthesis was elevated, indicating increased utilization of alanine. Creatine was unchanged in MRL/lpr mice as compared to controls. An interesting finding was a significant increase (158%, P<0.005) in choline concentration in MRL/lpr mice while the myo-inositol concentration remained the same in both groups. Furthermore a significant increase in total brain water content was observed, indicative of possible edema. In conclusion, the cumulative effect of increased brain lactate synthesis, altered glucose metabolism and intracellular glutamine accumulation could be an important mechanism causing brain pathology in SLE. The alteration in metabolites could alter downstream pathways and cause neurological dysfunction. Future NMR spectroscopic studies using stable isotopes and real-time measurements of metabolic rates, along with levels of metabolites in plasma and cerebrospinal fluid, could be valuable in the elucidation of the cerebral metabolic consequences of systemic lupus erythematosis (SLE) in humans.     

Glutathione-mediated Antioxidative Mechanisms in Sunflower (<Emphasis Type="Italic">Helianthus Annuus</Emphasis> L.) Cells in Response to Cadmium Stress

Cadmium (Cd) homeostasis and detoxification in sunflower (Helianthus annuus L.) cells differing in Cd sensitivity/tolerance were studied by analyzing the glutathione-mediated antioxidant mechanism vis-à-vis phytochelatin biosynthesis in vitro. Calluses exposed to Cd-shock/-acclimatization (150μM) were assayed for oxidative stress, reduced glutathione (GSH), glutathione disulfide (GSSG), phytochelatins (PCs) and reactive oxygen species (ROS). Although Cd did not induce any oxidative stress in Cd-tolerant callus (TCd), it generated oxidative stress in Cd-shock callus (SCd) both in terms of lipid peroxidation and protein oxidation. GSH/GSSG ratio remained similar to control values in the cadmium-acclimatized calluses. However, after acute treatment, there was a decline in both GSH and GSSG levels in SCd with concomitant reduction in the GSH/GSSG ratio. Analysis of PCs was performed using HPLC and mass spectrometry methods. PC concentration in TCd were approximately twice those that in SCd, showing in both cases a 1:2:1 relative proportion for PC n = 2 (PC2): PC n = 3 (PC3): PC n = 4 (PC4). Calluses growing in the presence of Cd developed an increased resistance to paraquat oxidative stress generation. These results indicated that PCs synthesis was an important mechanism for Cd detoxification in sunflower calluses, but the capacity to grow in the presence of Cd is related to the tissues ability to maintain high intracellular levels of GSH.     

Moderately thermophilic nitrifying bacteria from a hot spring of the Baikal rift zone

Samples from three hot springs (Alla, Seya and Garga) located in the northeastern part of Baikal rift zone (Buryat Republic, Russia) were screened for the presence of thermophilic nitrifying bacteria. Enrichment cultures were obtained solely from the Garga spring characterized by slightly alkaline water (pH 7.9) and an outlet temperature of 75 degrees C. The enrichment cultures of the ammonia- and nitrite oxidizers grew at temperature ranges of 27-55 and 40-60 degrees C, respectively. The temperature optimum was approximately 50 degrees C for both groups and thus they can be designated as moderate thermophiles. Ammonia oxidizers were identified with classical and immunological techniques. Representatives of the genus Nitrosomonas and Nitrosospira-like bacteria with characteristic vibroid morphology were detected. The latter were characterized by an enlarged periplasmic space, which has not been previously observed in ammonia oxidizers. Electron microscopy, denaturing gradient gel electrophoresis analyses and partial 16S rRNA gene sequencing provided evidence that the nitrite oxidizers were members of the genus Nitrospira.     

IL-6 expression induced by adenosine A2b receptor stimulation in U373 MG cells depends on p38 mitogen activated kinase and protein kinase C

Adenosine binds to a class of G-protein coupled receptors, which are further distinguished as A(1), A(2a), A(2b) and A(3) adenosine receptors. As we have shown earlier, the stable adenosine analogue NECA (N6-(R)-phenylisopropyladenosine) stimulates IL-6 expression in the human astrocytoma cell line U373 MG via the A(2b) receptor. The mechanism by which NECA promotes astrocytic IL-6 expression has not been identified. By using various inhibitors of signal transduction, we found that p38 mitogen-activated protein kinases (MAPK) activation (inhibitor SB202190), but not extracellular signal-regulated kinase (ERK) (PD98059) and c-jun N-terminal kinase (JNK)(SP600125), is essential in the NECA-induced signalling cascade that leads to the increase in IL-6 synthesis in U373 MG cells. Results obtained with protein kinase C (PKC) inhibitors that have different substrate specificities, indicated that the PKC delta and epsilon isoforms are also involved in adenosine receptor A(2b) dependent upregulation of IL-6 expression. This is supported by the fact that NECA induced the activation of PKC delta and epsilon in U373 MG cells.     

Volume regulation in cortical collecting duct cells: role of AQP2

BACKGROUND INFORMATION: The renal CCD (cortical collecting duct) plays a role in final volume and concentration of urine by a process that is regulated by the antidiuretic hormone, [arginine]vasopressin. This hormone induces an increase in water permeability due to the translocation of AQP2 (aquaporin 2) from the intracellular vesicles to the apical membrane of principal cells. During the transition from antidiuresis to diuresis, CCD cells are exposed to changes in environmental osmolality, and cell-volume regulation may be especially important for the maintenance of intracellular homoeostasis. Despite its importance, cell-volume regulation in CCD cells has not been widely investigated. Moreover, no studies have been carried out till date to evaluate the putative role of AQPs during this process in renal cells. RESULTS: In the present study, we have studied the regulatory cell-volume responses to hypo-osmotic or hyperosmotic challenges in two CCD cell lines: one not expressing AQPs and the other stably transfected with AQP2. We have used a fluorescent probe technique in which the acquisition of single-cell kinetic data can be simultaneously recorded with the intracellular pH. Experiments with hyperosmotic mannitol media demonstrated that, independent of AQP2 expression, CCD cells shrink but fail to show regulatory volume increase, at least under the studied conditions. In contrast, under hypo-osmotic shocks, regulatory volume decrease occurs and the activation of these mechanisms is more rapid in AQP2 transfected cells. This regulatory response takes place in parallel with intracellular acidification, which is faster in cells expressing AQP2. The acidification and the initial regulatory volume decrease response were inhibited by glibenclamide and BaCl2 only in AQP2 cells. CONCLUSIONS: These results suggest that increases in the osmotic water permeability due to the expression of AQP2 are critical for a rapid activation of regulatory volume decrease mechanisms, which would be linked to cystic fibrosis transmembrane conductance regulator and to barium-sensitive potassium channels.