Rates for repair of pBR 322 DNA radicals by thiols as measured by the gas explosion technique: evidence that counter-ion condensation and co-ion depletion are significant at physiological ionic strength.

Rates of rapair of pBR 322 plasmid DNA radicals by thiols of varying net charge (Z) at pH 7 and physiological ionic strength were measured using the oxygen explosion technique. The extent of conversion of supercoiled to relaxed circular plasmid was measured by HPLC as a function of the time of oxygen exposure before or after irradiation, the time-courses being fitted by a pseudo-first-order kinetic expression with k1 = k2[RSH]. Values of k2 (M-1 S-1) were: 2.1 x 10(5) (GSH, Z = -1), 1.4 x 10(6) (2-mercaptoethanol, Z = 0), 1.2 x 10(7) (cysteamine, Z = +1), 6.6 x 10(7) (WR-1065 or N-(2-mercaptoethyl)-1,3-diamino?? propane, Z = +2). The approximately 6-fold increase in rate with each unit increase in Z is attributed to concentration of cationic thiols near DNA as a consequence of counter-ion condensation and reduced levels of anionic thiols near DNA owing to co-ion depletion. The results are quantitatively consistent with chemical repair as a significant mechanism for radioprotection of cells by neutral and cationic thiols under aerobic conditions, but indicate that repair by GSH will compete effectively with oxygen only at low oxygen tension.

Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400 mg kg(-1)) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.

Water-compatible imprinted polymers for selective depletion of riboflavine from beverages

Artificial riboflavin receptors adapted to aqueous environments were studied for their ability to selectively extract riboflavine (Rf) from three types of beverages i.e. milk, beer and a multivitamin mixture. The basic receptor was first prepared by molecular imprinting in nonaqueous medium using a hydrogen-bond donor-acceptor-donor functional monomer (2,6-bis(acrylamido)pyridine), complementary to the imide motif of the template, riboflavin tetra-acetate as template and pentaerythritol triacrylate (PETA) as a hydrophilic cross-linking monomer. The polymer was then packed in columns and used for extraction of riboflavine from beverages. Riboflavine (Rf) was selectively removed from milk and an artificial vitamin mixture but the nonspecific binding was still significant, as judged from the binding of Rf to a control nonimprinted polymer. In order to suppress this nonspecific binding, attempts to hydrolytically hydrophilize the polymer matrix were performed. The preferred approach consisted in a controlled base hydrolysis of pendent unreacted acrylate groups, using hydroxides with differently sized counterions as reagents. This resulted in a decreased binding of Rf to both polymers, but to an equal extent implying a preferential suppression of the nonspecific contribution to the binding. The hydrophilized polymers, when subjected to beer, showed larger imprinting factors at lower phase ratios compared to the nontreated polymers and a maximum removal of 86% compared to 47% for the nonimprinted control polymer.

Depletion of the ubiquitin-binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr ΔF508 mutation improves the delivery of Burkholderia cenocepacia to the autophagic machinery

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ?F508 mutation is the most common. ?F508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ?F508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ?F508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ?F508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ?F508 macrophages.

Effect of short-term macrophage depletion in the development of posterior capsule opacification in rodents

Aim: To evaluate the role of macrophages in the development of posterior capsule opacification (PCO). Methods: For this purpose, an extracapsular lens extraction was performed in 18 consecutive Sprague-Dawley rats. Animals were treated with liposomal clodronate (Cl MDP-lip-treated group, n = 10) or phosphate-buffered saline (PBS) (control group, n = 8) 1 day preoperatively and on the first day postoperatively, and sacrificed 3 days postoperatively. Masked clinical, light microscopy and immunohistochemistry studies were conducted. The Fisher exact test and randomisation test were used to assess statistically differences between groups. Results: A statistically significant reduction in the number of macrophages (ED1+, ED7+, ED8+) was found in the Cl MDP-lip-treated group compared with the PBS-lip-treated group (p = 0.048, p = 0.004, p = 0.027, respectively). There were no statistically significant differences with regards to the presence/absence of central opacification (p = 0.29) and capsular wrinkling (p = 0.21) as detected clinically between groups. Similarly, a qualitative evaluation of the degree of PCO with regards to lens epithelial cell (LEC) proliferation, capsular wrinkling and Soemmerring ring formation showed no statistically significance between groups (p = 0.27, p = 0.061, p = 1.0, respectively). However, a statistically significant reduction in the number of lens epithelial cells (LEC) counted in the centre of the posterior capsule was found in the Cl MDP-lip- treated group (p = 0.009). Conclusion: Depletion of macrophages was accompanied by a reduction in LEC in the centre of the posterior capsule in rodents.

Source depletion and extent of melting in the Tongan sub-arc mantle

The fluid immobile High Field Strength Elements (HFSE) Nb and Ta can be used to distinguish between the effects of variable extents of melting and prior source depletion of the Tongan sub-arc mantle. Melting of spinel Iherzolite beneath the Lau Basin back-arc spreading centres has the ability to fractionate Nb from Ta due to the greater compatibility of the latter in clinopyroxene. The identified spatial variation in plate velocities and separation of melt extraction zones, combined with extremely depleted lavas make Tonga an ideal setting in which to test models for arc melt generation and the role of back-arc magmatism. We present new data acquired by laser ablation-ICPMS of fused sample glasses produced without the use of a melt fluxing agent. The results show an arc trend towards strongly sub-chondritic Nb/Ta (

ARTD1/PARP1 Negatively Regulates Glycolysis by Inhibiting Hexokinase 1 Independent of NAD(+) Depletion

ARTD1 (PARP1) is a key enzyme involved in DNA repair through the synthesis of poly(ADP-ribose) (PAR) in response to strand breaks, and it plays an important role in cell death following excessive DNA damage. ARTD1-induced cell death is associated with NAD(+) depletion and ATP loss; however, the molecular mechanism of ARTD1-mediated energy collapse remains elusive. Using real-time metabolic measurements, we compared the effects of ARTD1 activation and direct NAD(+) depletion. We found that ARTD1-mediated PAR synthesis, but not direct NAD(+) depletion, resulted in a block to glycolysis and ATP loss. We then established a proteomics-based PAR interactome after DNA damage and identified hexokinase 1 (HK1) as a PAR binding protein. HK1 activity is suppressed following nuclear ARTD1 activation and binding by PAR. These findings help explain how prolonged activation of ARTD1 triggers energy collapse and cell death, revealing insight into the importance of nucleus-to-mitochondria communication via ARTD1 activation.

A differential protein solubility approach for the depletion of highly abundant proteins in plasma using ammonium sulfate

Depletion of highly abundant proteins is an approved step in blood plasma analysis by mass spectrometry (MS). In this study, we explored a precipitation and differential protein solubility approach as a fractionation strategy for abundant protein removal from plasma. Total proteins from plasma were precipitated with 90% saturated ammonium sulfate, followed by differential solubilization in 55% and 35% saturated ammonium sulfate solutions. Using a four hour liquid chromatography (LC) gradient and an LTQ-Orbitrap XL mass spectrometer, a total of 167 and 224 proteins were identified from the 55% and 35% ammonium sulfate fractions, whereas 235 proteins were found in the remaining protein fractions with at least two unique peptides. SDS-PAGE and exclusive total spectrum counts from LC-MS/MS analyses clearly showed that majority of the abundant plasma proteins were solubilized in 55% and 35% ammonium sulfate solutions, indicating that the remaining protein fraction is of potential interest for identification of less abundant plasma proteins. Serum albumin, serotransferrin, alpha-1-antitrypsin and transthyretin were the abundant proteins that were highly enriched in 55% ammonium sulfate fractions. Immunoglobulins, complement system proteins, and apolipoproteins were among other abundant plasma proteins that were enriched in 35% ammonium sulfate fractions. In the remaining protein fractions a total of 40 unique proteins were identified of which, 32 proteins were identified with at least 10 exclusive spectrum counts. According to PeptideAtlas, 9 of these 32 proteins were estimated to be present at low μg ml(-1) (0.12-1.9 μg ml(-1)) concentrations in the plasma, and 17 at low ng ml(-1) (0.1-55 ng ml(-1)) range.