RdgB acts to avoid chromosome fragmentation in Escherichia coli

Bacterial RecA protein is required for repair of two-strand DNA lesions that disable whole chromosomes. recA mutants are viable, suggesting a considerable cellular capacity to avoid these chromosome-disabling lesions. recA-dependent mutants reveal chromosomal lesion avoidance pathways. Here we characterize one such mutant, rdgB/yggV, deficient in a putative inosine/xanthosine triphosphatase, conserved throughout kingdoms of life. The rdgB recA lethality is suppressed by inactivation of endonuclease V (gpnfi) specific for DNA-hypoxanthines/xanthines, suggesting that RdgB either intercepts improper DNA precursors dITP/dXTP or works downstream of EndoV in excision repair of incorporated hypoxathines/xanthines. We find that DNA isolated from rdgB mutants contains EndoV-recognizable modifications, whereas DNA from nfi mutants does not, substantiating the dITP/dXTP interception by RdgB. rdgB recBC cells are inviable, whereas rdgB recF cells are healthy, suggesting that chromosomes in rdgB mutants suffer double-strand breaks. Chromosomal fragmentation is indeed observed in rdgB recBC mutants and is suppressed in rdgB recBC nfi mutants. Thus, one way to avoid chromosomal lesions is to prevent hypoxanthine/xanthine incorporation into DNA via interception of dITP/dXTP.     

Stop codon recognition and interactions with peptide release factor RF3 of truncated and chimeric RF1 and RF2 from Escherichia coli

Release factors RF1 and RF2 recognize stop codons present at the A-site of the ribosome and activate hydrolysis of peptidyl-tRNA to release the peptide chain. Interactions with RF3, a ribosome-dependent GTPase, then initiate a series of reactions that accelerate the dissociation of RF1 or RF2 and their recycling between ribosomes. Two regions of Escherichia coli RF1 and RF2 were identified previously as involved in stop codon recognition and peptidyl-tRNA hydrolysis. We show here that removing the N-terminal domain of RF1 or RF2 or exchanging this domain between the two factors does not affect RF specificity but has different effects on the activity of RF1 and RF2: truncated RF1 remains highly active and able to support rapid cell growth, whereas cells with truncated RF2 grow only poorly. Transplanting a loop of 13 amino acid residues from RF2 to RF1 switches the stop codon specificity. The interaction of the truncated factors with RF3 on the ribosome is defective: they fail to stimulate guanine nucleotide exchange on RF3, recycling is not stimulated by RF3, and nucleotide-free RF3 fails to stabilize the binding of RF1 or RF2 to the ribosome. However, the N-terminal domain seems not to be required for the expulsion of RF1 or RF2 by RF3:GTP.     

Protection of Methanosarcina barkeri against oxidative stress: identification and characterization of an iron superoxide dismutase

Methanosarcina barkeri is a methanogenic archaeon that can only grow under strictly anoxic conditions but which can survive oxidative stress. We have recently reported that the organism contains a monofunctional catalase. We describe here that it also possesses an active iron superoxide dismutase. The enzyme was purified in three steps over 130-fold in a 14% yield to a specific activity of 1500 U/mg. SDS-PAGE revealed the presence of only one band, at an apparent molecular mass of 25 kDa. The primary structure determined from the cloned and sequenced gene revealed similarity to iron- and manganese superoxide dismutases. The highest similarity was to the iron superoxide dismutase from Methanobacterium thermoautotrophicum. The enzyme from M. barkeri was found to contain, per mol, 1 mol iron, but no manganese in agreement with the general observation that anaerobically growing organisms only contain iron superoxide dismutase. The enzyme was not inhibited by cyanide (10 mM), which is a property shared by all iron- and manganese superoxide dismutases. The presence of superoxide dismutase in M. barkeri is noteworthy since a gene encoding superoxide dismutase (sod) has not been found in Archaeoglobus fulgidus, a sulfate-reducing archaeon most closely related to the Methanosarcinaceae.     

Synthesis of the 15N-Gln11 labeled peptaibol antibiotic zervamicin-IIB

Summary Synthesis of zervamicin IIB, specifically labeled at the α-position of glutamine-11 with¹⁵N, was achieved by the Fmoc/tert.-butyl strategy in solution using a fragment condensation approach. Three fragments of zervamicin IIB were obtained by stepwise elongation with Fmoc amino acids using BOP as a coupling reagent. For the introduction of the highly sterically hindered α-aminoisobutyric acid residues, BOP/DMAP activation was applied. Peptide fragments were coupled by means of the coupling reagent, CF₃-PyBOP. Using the strategy developed, zervamicin IIB specifically¹⁵N labeled has been synthesized in 30% overall yield based on the isotopically labeled amino acid. From 600 MHz NMR spectroscopy the position of the¹⁵N-label was clearly detected. The isotope enrichment (98 ±2%) was determined by FAB-mass spectrometry.     

Dihydro-resveratrol—A potent dietary polyphenol

Dihydro-resveratrol (dihydro-R), a prominent polyphenol component of red wine, has a profound proliferative effect on hormone-sensitive tumor cell lines such as breast cancer cell line MCF7. We found a significant increase in MCF7 tumor cells growth rates in the presence of picomolar concentrations of this compound. The proliferative effect of dihydro-R was not observed in cell lines that do not express hormone receptors (MDA-MB-231, BT-474, and К-562).     

Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding β-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated β-catenin. By 2 months post fertilization (mpf), 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor) that suppressed this phenotype. We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated β-catenin. The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for β-catenin-induced liver tumors.     

Intraspecies Genomic Diversity and Long-Term Persistence of Bifidobacterium longum

Members of genus Bifidobacterium are Gram-positive bacteria, representing a large part of the human infant microbiota and moderately common in adults. However, our knowledge about their diversity, intraspecific phylogeny and long-term persistence in humans is still limited. Bifidobacterium longum is generally considered to be the most common and prevalent species in the intestinal microbiota. In this work we studied whole genome sequences of 28 strains of B. longum, including 8 sequences described in this paper. Part of these strains were isolated from healthy children during a long observation period (up to 10 years between isolation from the same patient). The three known subspecies (longum, infantis and suis) could be clearly divided using sequence-based phylogenetic methods, gene content and the average nucleotide identity. The profiles of glycoside hydrolase genes reflected the different ecological specializations of these three subspecies. The high impact of horizontal gene transfer on genomic diversity was observed, which is possibly due to a large number of prophages and rapidly spreading plasmids. The pan-genome characteristics of the subspecies longum corresponded to the open pan-genome model. While the major part of the strain-specific genetic loci represented transposons and phage-derived regions, a large number of cell envelope synthesis genes were also observed within this category, representing high variability of cell surface molecules. We observed the cases of isolation of high genetically similar strains of B. longum from the same patients after long periods of time, however, we didn’t succeed in the isolation of genetically identical bacteria: a fact, reflecting the high plasticity of microbiota in children.