Prevalence of vancomycin-resistant enterococci in hospitalized patients and those living in the community in the Czech Republic

Between July 1, 2002 and December 31, 2003, rectal swabs from both hospitalized patients and community subjects in the Czech Republic were taken to ascertain the prevalence of vancomycin-resistant enterococci (VRE). The swabs were used for isolating and identifying enterococci and their susceptibility to antibiotics. Vancomycin resistance phenotypes were verified by PCR detection of vanA, vanB, vanC1 and vanC2 genes. A molecular biology analysis was performed in Enterococcus faecium VanA strains. During the observed period, 2691 rectal swabs from the hospitalized patients and 6529 rectal swabs from the subjects in community setting were examined. In total, 31 VRE of hospital origin and 13 community-population strains were isolated. The prevalence of VRE in the gastrointestinal tract was 1.9% in the hospitalized patients and 0.4% in the community subjects. The prevailing strains were Enterococcus faecium VanA (61.3%) in the VRE of hospital origin and Enterococcus gallinarum VanC (46.2%) in the community VRE. Mutual comparison between the hospital and community Enterococcus faecium VanA strains showed no similarity.     

Claspin operates downstream of TopBP1 to direct ATR signaling towards Chk1 activation

TopBP1 and Claspin are adaptor proteins that facilitate phosphorylation of Chk1 by the ATR kinase in response to genotoxic stress. Despite their established requirement for Chk1 activation, the exact way in which TopBP1 and Claspin control Chk1 phosphorylation remains unclear. We show that TopBP1 tightly colocalizes with ATR in distinct nuclear subcompartments generated by DNA damage. Although depletion of TopBP1 by RNA interference (RNAi) strongly impaired phosphorylation of multiple ATR targets, including Chk1, Nbs1, Smc1, and H2AX, it did not interfere with ATR assembly at the sites of DNA damage. These findings challenge the current concept of ATR activation by recruitment to damaged DNA. In contrast, Claspin, like Chk1, remained distributed throughout the nucleus both before and after DNA damage. Consistently, the RNAi-mediated ablation of Claspin selectively abrogated ATR's ability to phosphorylate Chk1 but not other ATR targets. In addition, downregulation of Claspin mimicked Chk1 inactivation by inducing spontaneous DNA damage. Finally, we show that TopBP1 is required for the DNA damage-induced interaction between Claspin and Chk1. Together, these results suggest that while TopBP1 is a general regulator of ATR, Claspin operates downstream of TopBP1 to selectively regulate the Chk1-controlled branch of the genotoxic stress response.     

Structural and evolutionary classification of G/U wobble basepairs in the ribosome

We present a comprehensive structural, evolutionary and molecular dynamics (MD) study of the G/U wobble basepairs in the ribosome based on high-resolution crystal structures, including the recent Escherichia coli structure. These basepairs are classified according to their tertiary interactions, and sequence conservation at their positions is determined. G/U basepairs participating in tertiary interactions are more conserved than those lacking any interactions. Specific interactions occurring in the G/U shallow groove pocket--like packing interactions (P-interactions) and some phosphate backbone interactions (phosphate-in-pocket interactions)--lead to higher G/U conservation than others. Two salient cases of unique phylogenetic compensation are discovered. First, a P-interaction is conserved through a series of compensatory mutations involving all four participating nucleotides to preserve or restore the G/U in the optimal orientation. Second, a G/U basepair forming a P-interaction and another one forming a phosphate-in-pocket interaction are replaced by GNRA loops that maintain similar tertiary contacts. MD simulations were carried out on eight P-interactions. The specific GU/CG signature of this interaction observed in structure and sequence analysis was rationalized, and can now be used for improving sequence alignments.     

Alpha-tocopheryl succinate induces DR4 and DR5 expression by a p53-dependent route: implication for sensitisation of resistant cancer cells to TRAIL apoptosis

We evaluated the ability of alpha-tocopheryl succinate (alpha-TOS) to sensitise TRAIL-resistant malignant mesothelioma (MM) cells to TRAIL-induced apoptosis. We show that alpha-TOS activates expression of DR4/DR5 in a p53-dependent manner and re-establishes sensitivity of resistant MM cells to TRAIL-mediated apoptosis, as documented in p53wt MM cells but not in their p53null counterparts. MM cells selected for TRAIL resistance expressed low cell surface levels of DR4 and DR5. Treatment with sub-lethal doses of alpha-TOS restored expression of DR4 and DR5. The ability of alpha-TOS to modulate expression of pro-apoptotic genes may play a role in sensitisation of tumour cells to immunological stimuli.     

Sensing, threading, orienting, and cutting polymers with rigid-rod pores

This short review describes synthetic pores that are made from rigid-rod molecules and can bind oligo-and polymers such as polyacetylenes, p-oligophenyls, terpenoids, polypeptides, polysaccharides, and oligonucleotides. The spotlight is on recent breakthroughs to image the longtime elusive pore-polymer host-guest complexes as single giant pseudorotaxanes.     

ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks

It is generally thought that the DNA-damage checkpoint kinases, ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), work independently of one another. Here, we show that ATM and the nuclease activity of meiotic recombination 11 (Mre11) are required for the processing of DNA double-strand breaks (DSBs) to generate the replication protein A (RPA)-coated ssDNA that is needed for ATR recruitment and the subsequent phosphorylation and activation of Chk1. Moreover, we show that efficient ATM-dependent ATR activation in response to DSBs is restricted to the S and G2 cell cycle phases and requires CDK kinase activity. Thus, in response to DSBs, ATR activation is regulated by ATM in a cell-cycle dependent manner.     

Wild type N-ras displays anti-malignant properties, in part by downregulating decorin

Previously, we have shown that wild type N-ras (wt N-ras) harbors an anti-malignant effect against mutated Ras and in tumors without Ras mutations. To investigate the molecular bases of this anti-malignant activity, we have studied the potency of this anti-malignant effect in a model system against SV40 large T antigen (SV40T). We show that wild-type N-ras (wt N-ras) counteracts the effects of SV40T in NIH3T3 cells as seen by a decrease in proliferation, anchorage independence and changes in migration. We also show that wt N-ras elicits the same anti-malignant effects in some human tumor cell lines (HT1080 and MDA-MB-231). Through mRNA and microRNA (miRNAs) expression profiling we have identified genes (decorin) and miRNAs (mir-29A, let-7b) modulated by wt N-ras potentially responsible for the anti-malignant effect. Wt N-ras appears to mediate its anti-malignant effect by downregulating some of the targets of the TGFβ pathway and decorin, which are able to reverse the inhibition of migration induced by wt N-ras. Our experiments show that the molecules that mediate the anti-malignant effect by wt N-ras appear to be different from those modulated by transforming N-ras. The components of the pathways modulated by wt N-ras mediating its anti-malignant effects are potential targets for therapeutic intervention in cancer.