ERCC1 expression and RAD51B activity correlate with cell cycle response to platinum drug treatment not DNA repair

Background: The H69CIS200 and H69OX400 cell lines are novel models of low-level platinum-drug resistance. Resistance was not associated with increased cellular glutathione or decreased accumulation of platinum, rather the resistant cell lines have a cell cycle alteration allowing them to rapidly proliferate post drug treatment. Results: A decrease in ERCC1 protein expression and an increase in RAD51B foci activity was observed in association with the platinum induced cell cycle arrest but these changes did not correlate with resistance or altered DNA repair capacity. The H69 cells and resistant cell lines have a p53 mutation and consequently decrease expression of p21 in response to platinum drug treatment, promoting progression of the cell cycle instead of increasing p21 to maintain the arrest. Conclusion: Decreased ERCC1 protein and increased RAD51B foci may in part be mediating the maintenance of the cell cycle arrest in the sensitive cells. Resistance in the H69CIS200 and H69OX400 cells may therefore involve the regulation of ERCC1 and RAD51B independent of their roles in DNA repair. The novel mechanism of platinum resistance in the H69CIS200 and H69OX400 cells demonstrates the multifactorial nature of platinum resistance which can occur independently of alterations in DNA repair capacity and changes in ERCC1

Functional and Biogenetical Heterogeneity of the Inner Membrane of Rat-Liver Mitochondria

Rat liver mitochondria were fragmented by a combined technique of swelling, shrinking, and sonication. Fragments of inner membrane were separated by density gradient centrifugation. They differed in several respects: electronmicroscopic appearance, phospholipid and cytochrome contents, electrophoretic behaviour of proteins and enzymatic activities. Three types of inner membrane fractions were isolated. The first type is characterized by a high activity of metal chelatase, low activities of succinate-cytochrome c reductase and of glycerolphosphate dehydrogenase, as well as by a high phospholipid content and low contents of cytochromes aa3 and b. The second type displays maximal activities of glycerolphosphate dehydrogenase and metal chelatase, but contains relatively little cytochromes and has low succinate-cytochrome c reductase activity. The third type exhibits highest succinate-cytochrome c reductase activity, a high metal chelatase activity and highest cytochrome contents. However, this fraction was low in both glycerolphosphate dehydrogenase activity and phospholipid content. This fraction was also richest in the following enzyme activities: cytochrome oxidase, oligomycin-sensitive ATPase, proline oxidase, 3-hydroxybutyrate dehydrogenase and rotenone-sensitive NADH-cytochrome c reductase. Amino acid incorporation in vitro and in vivo in the presence of cycloheximide occurs predominantly into inner membrane fractions from the second type. These data suggest that the inner membrane is composed of differently organized parts, and that polypeptides synthesized by mitochondrial ribosomes are integrated into specific parts of the inner membrane

2, 4-Diamino-6- hydroxy pyrimidine inhibits NSAIDs induced nitrosyl-complex EPR signals and ulcer in rat jejunum

BACKGROUND: It has been suggested that one aspect of non-steroidal anti-inflammatory drugs induced intestinal damage is due to either uncoupling of mitochondrial oxidative phosphorylation or inhibition of electron transport. We investigated the latter possibility using electron paramagnetic resonance spectroscopy. RESULTS: Electron paramagnetic studies of NSAIDS on sub-mitochondrial particles revealed that indomethacin, but not with nabumetone, bound to a site near to Complex I and ubiquinone to generate a radical species. Normal rats exhibited prominent [3Fe-4S]ox signals (g ~ 2.01) at 20 K. One hour after indomethacin there was a prominent, intense and broad absorption pattern at (g ~2.07) suggesting, appearance of radical species overlapping [3Fe-4S]ox and was unaffected by pretreatment with 2,4 diamino -6-hydroxy pyrimidine. At 24 hrs, when macroscopic ulcers were seen, there was a new signal due to a nitric oxide radical (NO•). In contrast, nabumetone and 2,4 diamino-6-hydroxy pyrimidine pre-treated animals receiving indomethacin exhibited electron paramagnetic resonance spectra identical to those of controls at 24 hrs and neither was associated with small intestinal ulcers. Indomethacin and 2,4 diamino hydroxy pyrimidine pre-treated rats, but not nabumetone, had increased intestinal permeability. CONCLUSION: The results suggest that the in vivo effects of indomethacin modulate the mitochondrial respiratory chain directly at 1 h and 24 h through formation of nitric oxide. NO• appears to play an important role in the late pathogenic stages of NSAID enteropathy and may be the site for targeted treatment to reduce their toxicity

Disparate requirements for the Walker A and B ATPase motifs of human RAD51D in homologous recombination

In vertebrates, homologous recombinational repair (HRR) requires RAD51 and five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C and RAD51D) that all contain conserved Walker A and B ATPase motifs. In human RAD51D we examined the requirement for these motifs in interactions with XRCC2 and RAD51C, and for survival of cells in response to DNA interstrand crosslinks (ICLs). Ectopic expression of wild-type human RAD51D or mutants having a non-functional A or B motif was used to test for complementation of a rad51d knockout hamster CHO cell line. Although A-motif mutants complement very efficiently, B-motif mutants do not. Consistent with these results, experiments using the yeast two- and three-hybrid systems show that the interactions between RAD51D and its XRCC2 and RAD51C partners also require a functional RAD51D B motif, but not motif A. Similarly, hamster Xrcc2 is unable to bind to the non-complementing human RAD51D B-motif mutants in co-immunoprecipitation assays. We conclude that a functional Walker B motif, but not A motif, is necessary for RAD51D's interactions with other paralogs and for efficient HRR. We present a model in which ATPase sites are formed in a bipartite manner between RAD51D and other RAD51 paralogs

Effect of bilirubin on cytochrome c oxidase activity of mitochondria from mouse brain and liver

<p>Abstract</p> <p>Background</p> <p>The unbound, free concentration (B_f) of unconjugated bilirubin (UCB), and not the total UCB level, has been shown to correlate with bilirubin cytotoxicity, but the key molecular mechanisms accounting for the toxic effects of UCB are largely unknown.</p> <p>Findings</p> <p>Mouse liver mitochondria increase unbound UCB oxidation, consequently increasing the apparent rate constant for unbound UCB oxidation by HRP (Kp), higher than in control and mouse brain mitochondria, emphasizing the importance of determining Kp in complete systems containing the organelles being studied. The <it>in vitro </it>effects of UCB on cytochrome <it>c </it>oxidase activity in mitochondria isolated from mouse brain and liver were studied at B_franging from 22 to 150 nM. The results show that UCB at B_fup to 60 nM did not alter mitochondrial cytochrome <it>c </it>oxidase activity, while the higher concentrations significantly inhibited the enzyme activity by 20% in both liver and brain mitochondria.</p> <p>Conclusions</p> <p>We conclude that it is essential to include the organelles being studied in the medium used in measuring both Kp and B_f. A moderately elevated, pathophysiologically-relevant B_fimpaired the cytochrome <it>c </it>oxidase activity modestly in mitochondria from mouse brain and liver.</p

Redistribution of Actin during Assembly and Reassembly of the Contractile Ring in Grasshopper Spermatocytes

Cytokinesis in animal cells requires the assembly of an actomyosin contractile ring to cleave the cell. The ring is highly dynamic; it assembles and disassembles during each cell cleavage, resulting in the recurrent redistribution of actin. To investigate this process in grasshopper spermatocytes, we mechanically manipulated the spindle to induce actin redistribution into ectopic contractile rings, around reassembled lateral spindles. To enhance visualization of actin, we folded the spindle at its equator to convert the remnants of the partially assembled ring into a concentrated source of actin. Filaments from the disintegrating ring aligned along reorganizing spindle microtubules, suggesting that their incorporation into the new ring was mediated by microtubules. We tracked incorporation by speckling actin filaments with Qdots and/or labeling them with Alexa 488-phalloidin. The pattern of movement implied that actin was transported along spindle microtubules, before entering the ring. By double-labeling dividing cells, we imaged actin filaments moving along microtubules near the contractile ring. Together, our findings indicate that in one mechanism of actin redistribution, actin filaments are transported along spindle microtubule tracks in a plus-end–directed fashion. After reaching the spindle midzone, the filaments could be transported laterally to the ring. Notably, actin filaments undergo a dramatic trajectory change as they enter the ring, implying the existence of a pulling force. Two other mechanisms of actin redistribution, cortical flow and de novo assembly, are also present in grasshopper, suggesting that actin converges at the nascent contractile ring from diffuse sources within the cytoplasm and cortex, mediated by spindle microtubules

Homology modeling and molecular dynamics simulations of MUC1-9/H-2Kb complex suggest novel binding interactions

International audienceHuman MUC1 is over-expressed in human adenocarcinomas and has been used as a target for immunotherapy studies. The 9-mer MUC1-9 peptide has been identified as one of the peptides which binds to murine MHC class I H-2K. The structure of MUC1-9 in complex with H-2K has been modeled and simulated with classical molecular dynamics, based on the x-ray structure of the SEV9 peptide/H-2K complex. Two independent trajectories with the solvated complex (10 ns in length) were produced. Approximately 12 hydrogen bonds were identified during both trajectories to contribute to peptide/MHC complex, as well as 1-2 water mediated hydrogen bonds. Stability of the complex was also confirmed by buried surface area analysis, although the corresponding values were about 20% lower than those of the original x-ray structure. Interestingly, a bulged conformation of the peptide's central region, partially characterized as a -turn, was found exposed form the binding groove. In addition, P1 and P9 residues remained bound in the A and F binding pockets, even though there was a suggestion that P9 was more flexible. The complex lacked numerous water mediated hydrogen bonds that were present in the reference peptide x-ray structure. Moreover, local displacements of residues Asp4, Thr5 and Pro9 resulted in loss of some key interactions with the MHC molecule. This might explain the reduced affinity of the MUC1-9 peptide, relatively to SEV9, for the MHC class I H-2K

Extra-Renal Elimination of Uric Acid via Intestinal Efflux Transporter BCRP/ABCG2

Urinary excretion accounts for two-thirds of total elimination of uric acid and the remainder is excreted in feces. However, the mechanism of extra-renal elimination is poorly understood. In the present study, we aimed to clarify the mechanism and the extent of elimination of uric acid through liver and intestine using oxonate-treated rats and Caco-2 cells as a model of human intestinal epithelium. In oxonate-treated rats, significant amounts of externally administered and endogenous uric acid were recovered in the intestinal lumen, while biliary excretion was minimal. Accordingly, direct intestinal secretion was thought to be a substantial contributor to extra-renal elimination of uric acid. Since human efflux transporter BCRP/ABCG2 accepts uric acid as a substrate and genetic polymorphism causing a decrease of BCRP activity is known to be associated with hyperuricemia and gout, the contribution of rBcrp to intestinal secretion was examined. rBcrp was confirmed to transport uric acid in a membrane vesicle study, and intestinal regional differences of expression of rBcrp mRNA were well correlated with uric acid secretory activity into the intestinal lumen. Bcrp1 knockout mice exhibited significantly decreased intestinal secretion and an increased plasma concentration of uric acid. Furthermore, a Bcrp inhibitor, elacridar, caused a decrease of intestinal secretion of uric acid. In Caco-2 cells, uric acid showed a polarized flux from the basolateral to apical side, and this flux was almost abolished in the presence of elacridar. These results demonstrate that BCRP contributes at least in part to the intestinal excretion of uric acid as extra-renal elimination pathway in humans and rats

Atlantic cod (Gadus morhua) hemoglobin genes: multiplicity and polymorphism

Background: Hemoglobin (Hb) polymorphism, assessed by protein gel electrophoresis, has been used almost exclusively to characterize the genetic structure of Atlantic cod (Gadus morhua) populations and to establish correlations with phenotypic traits such as Hb oxygen binding capacity, temperature tolerance and growth characteristics. The genetic system used to explain the results of gel electrophoresis entails the presence of one polymorphic locus with two major alleles (HbI-1; HbI-2). However, vertebrates have more than one gene encoding Hbs and recent studies have reported that more than one Hb gene is present in Atlantic cod. These observations prompted us to re-evaluate the number of Hb genes expressed in Atlantic cod, and to perform an in depth search for polymorphisms that might produce relevant phenotypes for breeding programs. Results: Analysis of Expressed Sequence Tags (ESTs) led to the identification of nine distinct Hb transcripts; four corresponding to the α Hb gene family and five to the β Hb gene family. To gain insights about the Hb genes encoding these transcripts, genomic sequence data was generated from heterozygous (HbI-1/2) parents and fifteen progeny; five of each HbI type, i.e., HbI-1/1, HbI-1/2 and HbI-2/2. β Hb genes displayed more polymorphism than α Hb genes. Two major allele types (β1A and β1B) that differ by two linked non-synonymous substitutions (Met55Val and Lys62Ala) were found in the β1 Hb gene, and the distribution of these β1A and β1B alleles among individuals was congruent with that of the HbI-1 and HbI-2 alleles determined by protein gel electrophoresis. RT-PCR and Q-PCR analysis of the nine Hb genes indicates that all genes are expressed in adult fish, but their level of expression varies greatly; higher expression of almost all Hb genes was found in individuals displaying the HbI-2/2 electrophoretic type. Conclusion: This study indicates that more Hb genes are present and expressed in adult Atlantic cod than previously documented. Our finding that nine Hb genes are expressed simultaneously in adult fish suggests that Atlantic cod, similarly to fish such as rainbow trout, carp, and goldfish, might be able to respond to environmental challenges such as chronic hypoxia or long-term changes in temperature by altering the level of expression of these genes. In this context, the role of the non-conservative substitution Lys62Ala found in the β1 Hb gene, which appears to explain the occurrence of the HbI-1 and HbI-2 alleles described by gel electrophoresis, and which was found to be present in other fish such as eel, emerald rockcod, rainbow trout and moray, requires further investigation