Rad50 Is Dispensable for the Maintenance and Viability of Postmitotic Tissues

The majority of spontaneous chromosome breakage occurs during the process of DNA replication. Homologous recombination is the primary mechanism of repair of such damage, which probably accounts for the fact that it is essential for genome integrity and viability in mammalian cells. The Mre11 complex plays diverse roles in the maintenance of genomic integrity, influencing homologous recombination, checkpoint activation, and telomere maintenance. The complex is essential for cellular viability, but given its myriad influences on genomic integrity, the mechanistic basis for the nonviability of Mre11 complex-deficient cells has not been defined. In this study we generated mice carrying a conditional allele of Rad50 and examined the effects of Rad50 deficiency in proliferative and nonproliferative settings. Depletion of Rad50 in cultured cells caused extensive DNA damage and death within 3 to 5 days of Rad50 deletion. This was not associated with gross telomere dysfunction, suggesting that the telomeric functions of the Mre11 complex are not required for viability. Rad50 was also dispensable for the viability of quiescent liver and postmitotic Purkinje cells of the cerebellum. These findings support the idea that the essential functions of the Mre11 complex are associated with DNA replication and further suggest that homologous recombination is not essential in nondividing cells.The Mre11 complex regulates both DNA damage checkpoint function and repair. Its checkpoint functions appear to be primarily related to its role as a DNA double-strand break (DSB) sensor which binds DNA damage and activates ATM (ataxia-telangiectasia [AT] mutated). The ATM kinase transduces the damage signal via phosphorylating mediators of the damage response (30, 42), which promotes cell cycle arrest, DNA repair, and apoptosis. Mre11 complex functions are compromised in the human chromosome instability syndromes Nijmegen breakage syndrome and AT-like disorder, which are caused by hypomorphic mutations in Nbs1 and Mre11. Cells derived from patients and from mouse models of these diseases exhibit spontaneous DNA damage, ionizing radiation (IR) sensitivity, and checkpoint defects (25, 27, 48, 52, 57).The complex''s primary role in DNA repair is in recombinational DSB repair, and this role likely underlies its essential nature. In Saccharomyces cerevisiae, the complex governs homologous recombination (HR) and nonhomologous end joining (NHEJ) (19), whereas in vertebrate systems it primarily functions in HR (51, 61, 62). In fact, studies of Nbs1-deficient cells suggest that the Mre11 complex may inhibit NHEJ in mammals (62). Data from several species also implicate the Mre11 nuclease in the metabolism of topoisomerase adducts (40, 43, 49). This highly conserved function could also explain why the Mre11 complex is essential.The Mre11 complex''s function at telomeres may also be required for viability. Telomeres protect the ends of linear chromosomes from being recognized as DSBs and thereby activating the DNA damage response (DDR) (9). In S. cerevisiae the Mre11 complex influences telomere length maintenance (5, 28), whereas in mammals the complex interacts with the telomere binding protein Trf2 and localizes to telomeres (63). Loss of Trf2 results in telomere uncapping, causing activation of the DDR, telomere fusions, and senescence (7). Given the association of Mre11 with Trf2, it is conceivable that acute Mre11 complex deficiency in the mouse would phenocopy Trf2 loss and similarly lead to cell death as a result of telomere uncapping.Conclusions regarding the essential nature of HR in general (33, 47, 53) and the Mre11 complex specifically (10, 17, 45, 59, 62) have been derived from the analysis of proliferating cells in vitro or in vivo. The coincidence of DNA replication and the formation of spontaneous DSBs prompted us to test whether the Mre11 complex and, by extension, HR would be essential in quiescent or postmitotic tissues in which the frequency of spontaneous DSBs is significantly reduced. To examine this issue, we generated mice containing a conditional Rad50 allele in which the Rad50 gene could be inactivated in quiescent and postmitotic cells.Our results indicate that Rad50 is not required for homeostasis or viability of quiescent hepatocytes of the adult liver; nor does it appear to be required for maintenance of postmitotic Purkinje cells of the cerebellum. In contrast, Rad50 was required for viability of proliferating tissue culture and bone marrow cells. Rad50-deficient hepatocytes that were induced to divide via hepatectomy were able to achieve limited division and survived despite the presence of DNA damage that persisted long after the bulk of regeneration was complete. Rad50-deficient cells did not exhibit overtly dysfunctional telomeres, suggesting that their loss of viability was not due to acute telomere failure. These data indicate that the Mre11 complex and, by extension, HR may be dispensable in postmitotic cells and are consistent with the interpretation that the replication-associated functions of the Mre11 complex account for its essential nature.     

Evidence of indigenous NAH plasmid of naphthalene degrading Pseudomonas putida PpG7 strain implicated in limonin degradation

A well characterized naphthalene-degrading strain, Pseudomonas putida PpG7 was observed to utilize limonin, a highly-oxygenated triterpenoid compound as a sole source of carbon and energy. Limonin concentrations evidenced a 64% reduction over 48 h of growth in batch cultures. Attempts were made to acquire a plasmid-less derivative via various methods (viz. Ethidium Bromide, SDS, elevated temperature & mitomycin C), among which the method involving mitomycin C (20 ug/ml) proved successful. Concomitant with the loss of plasmid in P. putida PpG7 strain, the cured derivative was identified as a lim- phenotype. The lim+ phenotype could be conjugally transferred to the cured derivative. Based on the results of curing with mitomycin C, conjugation studies and presence of ndo gene encoding naphthalene 1,2 dioxygenase, it was demonstrated that genes for the limonin utilization were encoded on an 83 kb indigenous transmissible Inc. P9 NAH plasmid in Pseudomonas putida PpG7 strain.     

Chromatographic analysis of carbamazepine binding to human serum albumin. II. Comparison of the Schiff base and N-hydroxysuccinimide immobilization methods

Recent studies with carbamazepine on human serum albumin (HSA) columns have noted an appreciable degree of non-specific binding on supports prepared by the Schiff base immobilization method. This work examines an alternative immobilization method for HSA based on N-hydroxysuccinimide (NHS)-activated silica. This support was prepared by reacting HPLC-grade silica directly with disuccinimidyl carbonate. The resulting material was compared to an HSA support prepared by the Schiff base method in terms of its activity for carbamazepine and non-specific interactions with this drug. When examined by frontal analysis, both supports gave comparable association equilibrium constants for carbamazepine interactions with HSA ((0.53-0.55) x 10(4)M(-1) at 37 degrees C). However, columns prepared by the Schiff base method gave greater non-specific binding. These columns, as well as control columns prepared using the carbonyldiimidazole (CDI) immobilization method, were also evaluated for their non-specific binding to a variety of other solutes known to interact with HSA. From these results it was concluded that the NHS method was an attractive alternative to the Schiff base technique in the preparation of immobilized HSA for HPLC-based binding studies for carbamazepine. However, it was also noted that non-specific binding varies from one drug to the next in these immobilization methods, indicating that such properties should be evaluated on a case-by-case basis in the use and development of HSA columns for binding studies.     

Migration defects by DISC1 knockdown in C57BL/6, 129X1/SvJ, and ICR strains via in utero gene transfer and virus-mediated RNAi

Disrupted-in-Schizophrenia 1 (DISC1) is a promising genetic risk factor for major mental disorders. Many groups repeatedly reported a role for DISC1 in brain development in various strains of mice and rats by using RNA interference (RNAi) approach. Nonetheless, due to the complexity of its molecular disposition, such as many splice variants and a spontaneous deletion in a coding exon of the DISC1 gene in some mouse strains, there have been debates on the interpretation on these published data. Thus, in this study, we address this question by DISC1 knockdown via short-hairpin RNAs (shRNAs) against several distinct target sequences with more than one delivery methodologies into several mouse strains, including C57BL/6, ICR, and 129X1/SvJ. Here, we show that DISC1 knockdown by in utero electroporation of shRNA against exons 2, 6, and 10 consistently results in neuronal migration defects in the developing cerebral cortex, which are successfully rescued by co-expression of full-length DISC1. Furthermore, lentivirus-mediated shRNA also led to migration defects, which is consistent with two other methodologies already published, such as plasmid-mediated and retrovirus-mediated ones. The previous study by Song’s group also reported that, in the adult hippocampus, the phenotype elicited by DISC1 knockdown with shRNA targeting exon 2 was consistently seen in both C57BL/6 and 129S6 mice. Taken together, we propose that some of DISC1 isoforms that are feasible to be knocked down by shRNAs to exon 2, 6, and 10 of the DISC1 gene play a key role for neuronal migration commonly in various mouse strains and rats.     

Comparison of SYPRO Ruby and Flamingo fluorescent stains for application in proteomic research

Fluorescent dyes are widely used for the detection and quantitation of proteins separated by polyacrylamide gel electrophoresis. SYPRO Ruby is one such fluorescent dye widely used for this purpose. More recently, another fluorescent dye, Flamingo, is available for expression proteomic research. Using a standard ultraviolet (UV) transilluminator and a charge-coupled device (CCD)-based imaging system, the relative sensitivity of these two different fluorescent stains with regard to detection of protein spots separated by two-dimensional gel electrophoresis (2D-GE) and identification by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) were compared. Using mouse kidney and liver homogenates as well as Escherichia coli extract, we detected a greater number of protein spots using Flamingo compared with SYPRO Ruby. In addition, when we compared the number of matched peptides and the percentage of amino acid residues identified for 22 different protein spots of mouse kidney proteome, we observed a higher number of matched peptides and a higher percentage of amino acid residues for the majority of the proteins using Flamingo compared with SYPRO Ruby. Also, we were able to characterize a protein spot that can be detected by Flamingo only. Therefore, we recommend Flamingo over SYPRO Ruby to be used for studies on expression proteomics.     

Plant species traits across a riparian‐zone/forest ecotone

Abstract. We examined the changes in prevalence of nine plant traits – including the presence of woody stem tissue, leaf longevity, nitrogen fixation, seed longevity, dispersal vector, pollination vector, and clonal growth form – across a riparian/forest‐understory ecotone. This ecotone, found along headwater streams in boreal mixed‐wood forests, supports four distinct vegetation zones: streambank, riparian, transition, and upland forest understory. The objective of this study was to identify specific trait patterns that may indicate functional responses to the changes in environmental factors such as nutrient availability and wind exposure that occur across the ecotone. The suites of plant species traits found in each zone were distinct, with a strong change in the prevalence of several traits. Wind and insect pollination, wind and vertebrate diaspore dispersal, and deciduous and evergreen leaves showed the greatest change in prevalence between the vegetation types. Some traits, including insect pollination and vertebrate diaspore dispersal, were strongly correlated within species. The consistent cooccurrence of pairs of traits in the same species suggests common responses by very different traits to the same environmental factor. This study demonstrates that an ecotone can be characterized not only as a discontinuity in species distributions or environmental factors, but also as a discontinuity in the trait spectrum. Examining ecotones from a trait perspective has strong potential for identifying the environmental factors and associated species functional responses that encourage the development of distinct vegetation boundaries.     

Insight into the location and dynamics of the annexin A2 N-terminal domain during Ca-induced membrane bridging

Annexin A2 (AnxA2) is a Ca²⁺- and phospholipid-binding protein involved in many cellular regulatory processes. Like other annexins, it is constituted by two domains: a conserved core, containing the Ca²⁺ binding sites, and a variable N-terminal segment, containing sites for interactions with other protein partners like S100A10 (p11). A wealth of data exists on the structure and dynamics of the core, but little is known about the N-terminal domain especially in the Ca²⁺-induced membrane-bridging process. To investigate this protein region in the monomeric AnxA2 and in the heterotetramer (AnxA2-p11)₂, the reactive Cys8 residue was specifically labelled with the fluorescent probe acrylodan and the interactions with membranes were studied by steady-state and time-resolved fluorescence. In membrane junctions formed by the (AnxA2-p11)₂ heterotetramer, the flexibility of the N-terminal domain increased as compared to the protein in solution. In “homotypic” membrane junctions formed by monomeric AnxA2, acrylodan moved to a more hydrophobic environment than in the protein in solution and the flexibility of the N-terminal domain also increased. In these junctions, this domain is probably not in close contact with the membrane surface, as suggested by the weak quenching of acrylodan observed with doxyl-PCs, but pairs of N-termini likely interact, as revealed by the excimer-forming probe pyrene-maleimide bound to Cys8. We present a model of monomeric AnxA2 N-terminal domain organization in “homotypic” bridged membranes in the presence of Ca²⁺.     

Computational studies of CXCR1, the receptor of IL-8/CXCL8, using molecular dynamics and electrostatics

The three-dimensional structure of IL-8/CXCL8 has been previously determined using NMR spectroscopy and X-ray crystallography, but the structure of the receptors for this chemokine has not been determined experimentally. We present here the development of a model for the structure of the IL-8/CXCL8 receptor CXCR1, using a combination of homology modeling and a molecular dynamics simulation. Based on this model, we discuss the analysis of structural, dynamic, and physicochemical properties of CXCR1. We focused on the role of pairwise ionic interactions in local structural stability of CXCR1 and the role of electrostatic potentials in recognition of CXCR1 with IL-8/CXCL8. We have performed theoretical mutations of six charged amino acids in CXCR1, which abolish binding as suggested by earlier experimental data, to shed light on the effect of charge on association ability. We propose that the observed loss of binding in the six CXCR1 mutants is owed to loss of local structural stability, rather than hindrance of the recognition process because of changes in the overall electrostatic properties of the receptor. Based on further structural analysis, we propose some mutations of charged residues involving ion pairs in different elements of transmembrane helices and extracellular loops, which are expected to alter the local structure and possibly affect binding.