Induction and processing of complex DNA damage in human breast cancer cells MCF-7 and nonmalignant MCF-10A cells

Oxidatively induced stress and DNA damage have been associated with various human pathophysiological conditions, including cancer and aging. Complex DNA damage such as double-strand breaks (DSBs) and non-DSB bistranded oxidatively induced clustered DNA lesions (OCDL) (two or more DNA lesions within a short DNA fragment of 1-10 bp on opposing DNA strands) are hypothesized to be repair-resistant lesions challenging the repair mechanisms of the cell. To evaluate the induction and processing of complex DNA damage in breast cancer cells exposed to radiotherapy-relevant gamma-ray doses, we measured single-strand breaks (SSBs), DSBs, and OCDL in MCF-7 and HCC1937 malignant cells as well as MCF-10A nonmalignant human breast cells. For the detection and measurement of SSBs, DSBs, and OCDL, we used the alkaline single-cell gel electrophoresis, gamma-H2AX assay, and an adaptation of pulsed-field gel electrophoresis with E. coli repair enzymes as DNA damage probes. Increased levels for most types of DNA damage were detected in MCF-7 cells while the processing of DSBs and OCDL was deficient in these cells compared to MCF-10A cells. Furthermore, the total antioxidant capacity of MCF-7 cells was lower compared to their nonmalignant counterparts. These findings point to the important role of complex DNA damage in breast cancer and its potential association with breast cancer development especially in the case of deficient BRCA1 expression.     

A novel monoacylglycerol lipase inhibitor with analgesic and anti-inflammatory activity

A variety of long chain 1,2-diamines and related compounds were synthesized and tested for their activity on fatty acid amide hydrolase (FAAH) and monoacyglycerol lipase (MGL). (2S,9Z)-Octadec-9-ene-1,2-diamine selectively inhibits MGL (K(i) 21.8 microM) without significantly affecting FAAH. This compound exhibited interesting in vivo analgesic and anti-inflammatory properties, suggesting that selective inhibitors of MGL may be valuable novel agents for the treatment of inflammatory pain.     

Efficient computation of the phylogenetic likelihood function on multi-gene alignments and multi-core architectures

The continuous accumulation of sequence data, for example, due to novel wet-laboratory techniques such as pyrosequencing, coupled with the increasing popularity of multi-gene phylogenies and emerging multi-core processor architectures that face problems of cache congestion, poses new challenges with respect to the efficient computation of the phylogenetic maximum-likelihood (ML) function. Here, we propose two approaches that can significantly speed up likelihood computations that typically represent over 95 per cent of the computational effort conducted by current ML or Bayesian inference programs. Initially, we present a method and an appropriate data structure to efficiently compute the likelihood score on 'gappy' multi-gene alignments. By 'gappy' we denote sampling-induced gaps owing to missing sequences in individual genes (partitions), i.e. not real alignment gaps. A first proof-of-concept implementation in RAXML indicates that this approach can accelerate inferences on large and gappy alignments by approximately one order of magnitude. Moreover, we present insights and initial performance results on multi-core architectures obtained during the transition from an OpenMP-based to a Pthreads-based fine-grained parallelization of the ML function.     

Bacterial Deterioration of Marble Monuments: A Case Study of the Conservation Project of Acropolis Monuments

The deterioration of the Acropolis monuments’ surface can be attributed to a combination of mechanical, physical, chemical and biological factors. The aim of this work was to investigate the potential use of biocides suitable for marble surface in order to confront the biodeterioration phenomenon due to heterotrophic bacteria. Surface bacterial populations were studied in respect to the application of three biocide agents. A significant decrease of the bacterial counts was occurred after the biocides’ application and remained less compared with the counts occurred on the untreated surfaces for more than 14 months. A gradual diminution of bacterial diversity was also observed leading to the survival of specific Gram positive strains.     

Assembly,heterogeneity, and breaching of the basement membranes

Basement membranes are thin sheets of self-assembled extracellular matrices that are essential for embryonic development and for the homeostasis of adult tissues. They play a role in structuring, protecting, polarizing, and compartmentalizing cells, as well as in supplying them with growth factors. All basement membranes are built from laminin and collagen IV networks stabilized by nidogen/perlecan bridges. The precise composition of basement membranes, however, varies between different tissues. Even though basement membranes represent physical barriers that delimit different tissues, they are breached in many physiological or pathological processes, including development, the immune response, and tumor invasion. Here, we provide a brief overview of the molecular composition of basement membranes and the process of their assembly. We will then illustrate the heterogeneity of basement membranes using two examples, the epithelial basement membrane in the gut and the vascular basement membrane. Finally, we examine the different strategies cells use to breach the basement membrane.     

Does fragmented river connectivity alter the reproductive behavior of the potamodromous fish Alburnus vistonicus?

Blocked riverine spawning migration routes constitute one of the main threats of potamodromous fish that require well-connected habitats of high quality to maintain their unique life-history form. Alburnus vistonicus, a potamodromous species inhabiting the Vistonis freshwater system in northern Greece, manages to cope with wide salinity fluctuations in Vistonis Lake, but the impact of the fragmented connectivity of the lake’s two main freshwater suppliers, Kosynthos and Kompsatos rivers, has not been assessed. We analyzed the species whole reproductive cycle and we divided it into phases. Cyclicity was observed in gonadosomatic index and oocyte diameter, while total fecundity remained constant prior to the onset of the spawning season. Stomach fullness and condition factor differed among the surveyed habitats. Successful spawning and multiple spawning were verified, as well as spawning migration and potamodromy. However, mature females with markers of recent spawning activity were captured also in the lake, suggesting spawning occurrence in the lake or a previously undocumented oscillatory behavior between the lake and the rivers during the spawning season and between sequential spawning events. Either behavior may serve as a mechanism to overcome the restricted spawning migration routes due to the fragmented river connectivity.

     

13-Methylarachidonic Acid Is a Positive Allosteric Modulator of Endocannabinoid Oxygenation by Cyclooxygenase

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing k_cat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism.     

Changes in the conformation of 5S rRNA cause alterations in principal functions of the ribosomal nanomachine

5S rRNA is an integral component of the large ribosomal subunit in virtually all living organisms. Polyamine binding to 5S rRNA was investigated by cross-linking of N¹-azidobenzamidino (ABA)-spermine to naked 5S rRNA or 50S ribosomal subunits and whole ribosomes from Escherichia coli cells. ABA-spermine cross-linking sites were kinetically measured and their positions in 5S rRNA were localized by primer extension analysis. Helices III and V, and loops A, C, D and E in naked 5S rRNA were found to be preferred polyamine binding sites. When 50S ribosomal subunits or poly(U)-programmed 70S ribosomes bearing tRNA^Phe at the E-site and AcPhe-tRNA at the P-site were targeted, the susceptibility of 5S rRNA to ABA-spermine was greatly reduced. Regardless of 5S rRNA assembly status, binding of spermine induced significant changes in the 5S rRNA conformation; loop A adopted an apparent ‘loosening’ of its structure, while loops C, D, E and helices III and V achieved a more compact folding. Poly(U)-programmed 70S ribosomes possessing 5S rRNA cross-linked with spermine were more efficient than control ribosomes in tRNA binding, peptidyl transferase activity and translocation. Our results support the notion that 5S rRNA serves as a signal transducer between regions of 23S rRNA responsible for principal ribosomal functions.