Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2

The Chk2 Ser/Thr kinase plays crucial, evolutionarily conserved roles in cellular responses to DNA damage. Identification of two pro-oncogenic mutations within the Chk2 FHA domain has highlighted its importance for Chk2 function in checkpoint activation. The X-ray structure of the Chk2 FHA domain in complex with an in vitro selected phosphopeptide motif reveals the determinants of binding specificity and shows that both mutations are remote from the peptide binding site. We show that the Chk2 FHA domain mediates ATM-dependent Chk2 phosphorylation and targeting of Chk2 to in vivo binding partners such as BRCA1 through either or both of two structurally distinct mechanisms. Although phospho-dependent binding is important for Chk2 activity, previously uncharacterized phospho-independent FHA domain interactions appear to be the primary target of oncogenic lesions.     

A critique of widely used normalization software tools and an alternative method to identify reliable reference genes in red clover (Trifolium pratense L.)

Determination of appropriate reference genes is crucial to normalization of gene expression data and prevention of biased results in qRT-PCR studies. This study is the first attempt to systematically compare potential reference genes to detect the most constitutively expressed reference genes for accurate normalization in red clover tissues including leaves, stems and roots. To identify the best-suited reference gene(s) for normalization, several statistical algorithms such as geNorm, BestKeeper and NormFinder have been developed. All these algorithms are based on the key assumption that none of the investigated candidate reference genes show systematic variation in their expression profile across the samples being considered. However, this assumption is likely to be violated in practice. The authors therefore suggest a simple and novel stability index based on the analysis of variance model which is free from the assumption made by the algorithms. We assessed the expression stability of eight candidate reference genes including actin (ACT), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), elongation factor-1alpha (EF-1α), translation initiation factor (EIF-4a), ubiquitin-conjugating enzyme E2 (UBC2), polyubiquitin (UBQ10), sand family protein (SAND) and yellow-leaf-specific protein 8 (YLS8). Our results indicated that UBC2 and UBQ10 ranked as the two most stably expressed genes in leaf tissue. UBC2 and YLS8 were defined as optimal control genes for stem tissue. EIF-4a and UBC2 were found to be the most stable reference gene for root tissue. GAPDH and SAND showed relatively low stability in expression study of red clover. When all tested tissues were considered, we observed that YLS8 and UBC2 showed remarkable stability in their expression level across tissues.     

Agglomerates of aberrant DNA methylation are associated with toxicant-induced malignant transformation

Epigenetic dysfunction is a known contributor in carcinogenesis, and is emerging as a mechanism involved in toxicant-induced malignant transformation for environmental carcinogens such as arsenicals or cadmium. In addition to aberrant DNA methylation of single genes, another manifestation of epigenetic dysfunction in cancer is agglomerative DNA methylation, which can participate in long-range epigenetic silencing that targets many neighboring genes and has been shown to occur in several types of clinical cancers. Using in vitro model systems of toxicant-induced malignant transformation, we found hundreds of aberrant DNA methylation events that emerge during malignant transformation, some of which occur in an agglomerative fashion. In an arsenite-transformed prostate epithelial cell line, the protocadherin (PCDH), HOXC and HOXD gene family clusters are targeted for agglomerative DNA methylation. The agglomerative DNA methylation changes induced by arsenicals appear to be common and clinically relevant events, since they occur in other human cancer cell lines and models of malignant transformation, as well as clinical cancer specimens. Aberrant DNA methylation in general occurred more often within histone H3 lysine-27 trimethylation stem cell domains. We found a striking association between enrichment of histone H3 lysine-9 trimethylation stem cell domains and toxicant-induced agglomerative DNA methylation, suggesting these epigenetic modifications may become aberrantly linked during malignant transformation. In summary, we found an association between toxicant-induced malignant transformation and agglomerative DNA methylation, which lends further support to the hypothesis that epigenetic dysfunction plays an important role in toxicant-induced malignant transformation.     

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

Use of 16S-23S rRNA spacer-region (SR)-PCR for identification of intestinal clostridia

The suitability of a species identification technique based on PCR analysis of 16S-23S rRNA spacer region (SR) polymorphism for human intestinal Clostridium species was evaluated. This SR-PCR based technique is highly reproducible and successfully differentiated the strains tested, which included 17 ATCC type strains of Clostridium and 152 human stool Clostridium isolates, at the species or intraspecies level. Ninety-eight of 152 stool isolates, including C. bifermentans, C. butyricum, C. cadaveris, C. orbiscindens, C. paraputrificum, C. pefringens, C. ramosum, C. scindens, C. spiroforme, C. symbiosum and C. tertium, were identified to species level by SR-PCR patterns that were identical to those of their corresponding ATCC type strains. The other 54 stool isolates distributed among ten SR-PCR patterns that are unique and possibly represent ten novel Clostridium species or subspecies. The species identification obtained by SR-PCR pattern analysis completely agreed with that obtained by 16S rRNA sequencing, and led to identification that clearly differed from that obtained by cellular fatty acid analysis for 23/152 strains (15%). These results indicate that SR-PCR provides an accurate and rapid molecular method for the identification of human intestinal Clostridium species.     

Association of the M blood group system with bovine mastitis

Associations of the 11 bovine blood group systems with mastitis were examined in Red Danish dairy cattle. The mastitis status was followed during three lactational periods. A significant effect of the M blood group system on mastitis incidence was observed in the first and second lactation periods and a lower frequency of mastitis is found among animals lacking the M' factor as compared to those having the M' blood group factor. The significance of these results are discussed in view of the close relation between the M blood group system and the bovine lymphocyte antigens (BoLA), and the expected effect of eliminating the M' gene from the breed is estimated. Among the remaining 10 blood group systems, the T' system was the only system showing an overall effect on mastitis, and only in first and third lactation. However, the T' system was inconsistent with regard to the effect of the T' gene on the various mastitis diagnoses.