Expanding the Direct HetR Regulon in Anabaena sp. Strain PCC 7120

In response to a lack of environmental combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 differentiates nitrogen-fixing heterocyst cells in a periodic pattern. HetR is a transcription factor that coordinates the regulation of this developmental program. An inverted repeat-containing sequence in the hepA promoter required for proheterocyst-specific transcription was identified based on sequence similarity to a previously characterized binding site for HetR in the promoter of hetP. The binding affinity of HetR for the hepA site is roughly an order of magnitude lower than that for the hetP binding site. A BLAST search of the Anabaena genome identified 166 hepA-like sites that occur as single or tandem sites (two binding sites separated by 13 bp). The vast majority of these sites are present in predicted intergenic regions. HetR bound five representative single binding sites in vitro, and binding was abrogated by transversions in the binding sites that conserved the inverted repeat nature of the sites. Binding to four representative tandem sites was not observed. Transcriptional fusions of the green fluorescent protein gene gfp with putative promoter regions associated with the representative binding sites indicated that HetR could function as either an activator or repressor and that activation was cell-type specific. Taken together, we have expanded the direct HetR regulon and propose a model in which three categories of HetR binding sites, based on binding affinity and nucleotide sequence, contribute to three of the four phases of differentiation

Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

Peer reviewe

Evidence of Disseminated Intravascular Coagulation in a Hemorrhagic Fever with Renal Syndrome—Scoring Models and Severe Illness

Background: Viral hemorrhagic fevers (VHF) are considered to be a serious threat to public health worldwide with up to 100 million cases annually. The general hypothesis is that disseminated intravascular coagulation (DIC) is an important part of the pathogenesis. The study objectives were to study the variability of DIC in consecutive patients with acute hemorrhagic fever with renal syndrome (HFRS), and to evaluate if different established DIC-scores can be used as a prognostic marker for a more severe illness. Method and Findings: In a prospective study 2006–2008, data from 106 patients with confirmed HFRS were analyzed and scored for the presence of DIC according to six different templates based on criteria from the International Society on Thrombosis and Haemostasis (ISTH). The DIC-scoring templates with a fibrinogen/CRP-ratio were most predictive, with predictions for moderate/severe illness (p,0.01) and bleeding of moderate/major importance (p,0.05). With these templates, 18.9–28.3 % of the patients were diagnosed with DIC. Conclusions: DIC was found in about one fourth of the patients and correlated with a more severe disease. This supports that DIC is an important part of the pathogenesis in HFRS. ISTH-scores including fibrinogen/CRP-ratio outperform models without. The high negative predictive value could be a valuable tool for the clinician. We also believe that our findings coul

The DNA-Binding Domain of Yeast Rap1 Interacts with Double-Stranded DNA in Multiple Binding Modes

<i>Saccharomyces cerevisiae</i> repressor-activator protein 1 (Rap1) is an essential protein involved in multiple steps of DNA regulation, as an activator in transcription, as a repressor at silencer elements, and as a major component of the shelterin-like complex at telomeres. All the known functions of Rap1 require the known high-affinity and specific interaction of the DNA-binding domain with its recognition sequences. In this work, we focus on the interaction of the DNA-binding domain of Rap1 (Rap1^DBD) with double-stranded DNA substrates. Unexpectedly, we found that while Rap1^DBD forms a high-affinity 1:1 complex with its DNA recognition site, it can also form lower-affinity complexes with higher stoichiometries on DNA. These lower-affinity interactions are independent of the presence of the recognition sequence, and we propose they originate from the ability of Rap1^DBD to bind to DNA in two different binding modes. In one high-affinity binding mode, Rap1^DBD likely binds in the conformation observed in the available crystal structures. In the other alternative lower-affinity binding mode, we propose that a single Myb-like domain of the Rap1^DBD makes interactions with DNA, allowing for more than one protein molecule to bind to the DNA substrates. Our findings suggest that the Rap1^DBD does not simply target the protein to its recognition sequence but rather it might be a possible point of regulation

N-Sulfonyl Dipeptide Nitriles as Inhibitors of Human Cathepsin S: In silico Design, Synthesis and Biochemical Characterization

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (Ki = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4′-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (Ki = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S

Repression of the miR-143/145 cluster by oncogenic Ras initiates a tumor-promoting feed-forward pathway

Although activating mutations in RAS oncogenes are known to result in aberrant signaling through multiple pathways, the role of microRNAs (miRNAs) in the Ras oncogenic program remains poorly characterized. Here we demonstrate that Ras activation leads to repression of the miR-143/145 cluster in cells of human, murine, and zebrafish origin. Loss of miR-143/145 expression is observed frequently in KRAS mutant pancreatic cancers, and restoration of these miRNAs abrogates tumorigenesis. miR-143/145 down-regulation requires the Ras-responsive element-binding protein (RREB1), which represses the miR-143/145 promoter. Additionally, KRAS and RREB1 are targets of miR-143/miR-145, revealing a feed-forward mechanism that potentiates Ras signaling

Correction: Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses.

[This corrects the article DOI: 10.1371/journal.pntd.0002157.]