NADPH Oxidase-generated Hydrogen Peroxide Induces DNA Damage in Mutant FLT3-expressing Leukemia Cells

Internal tandem duplication of the FMS-like tyrosine kinase (FLT3-ITD) receptor is present in 20% of acute myeloid leukemia (AML) patients and it has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (DSBs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Our group has previously reported that inhibition of FLT3-ITD signaling results in post-translational down-regulation of p22^phox, a small membrane-bound subunit of the NADPH oxidase (NOX) complex. Here we demonstrated that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, have a higher protein level of p22^phox and p22^phox-interacting NOX isoforms than 32D cells transfected with the wild type FLT3 receptor (FLT3-WT). The inhibition of NOX proteins, p22^phox, and NOX protein knockdowns caused a reduction in ROS, as measured with a hydrogen peroxide (H₂O₂)-specific dye, peroxy orange 1 (PO1), and nuclear H₂O₂, as measured with nuclear peroxy emerald 1 (NucPE1). These reductions in the level of H₂O₂ following the NOX knockdowns were accompanied by a decrease in the number of DNA DSBs. We showed that 32D cells that express FLT3-ITD have a higher level of both oxidized DNA and DNA DSBs than their wild type counterparts. We also observed that NOX4 and p22^phox localize to the nuclear membrane in MV4–11 cells expressing FLT3-ITD. Taken together these data indicate that NOX and p22^phox mediate the ROS production from FLT3-ITD that signal to the nucleus causing genomic instability.     

The impact of nisin on sensitive and resistant mutants of Listeria monocytogenes in cottage cheese

Aims: Listeria monocytogenesΔgadD1 and ΔlisK mutants display enhanced and reduced sensitivity, respectively, to the food preservative nisin in laboratory media. However, the behaviour of these strains in a nisin‐containing food has not been assessed. Here we use cottage cheese as a model food to address this issue. Materials and Results: Antibiotic‐resistant forms of the wild‐type and mutant strains were employed to investigate the behaviour of multiple strains in a single food sample, thereby eliminating the problem of intersample variation. Using this approach, it was established that percentage survival of the ΔlisK mutant was greater than the parent strain in the absence of nisin and that this relative difference became even more dramatic in cottage cheese supplemented with nisin. The numbers of the ΔgadD1 mutant decreased more rapidly than the parent in cottage cheese without nisin, but surprisingly this trend was reversed in nisin‐supplemented cheese. Upon the addition of 10 mmol l^?1 monosodium glutamate, a substrate for the glutamate decarboxylase (GAD) system, the wild‐type LO28 strain regained its relative advantage over ΔgadD1. Conclusions: Care needs to be taken when predicting the behaviour of mutants of L. monocytogenes with altered resistance to nisin in food as experiments in laboratory media are not always a good indicator of how the strains will behave in such food environments. Significance and impact of the Study: This study further emphasizes the importance of utilizing food matrices to confirm observations made using laboratory media.     

Further Identification of Novel Lantibiotic Operons Using LanM-Based Genome Mining

Lantibiotics are gene-encoded antimicrobial peptides that are distinguished by the presence of the unusual structures, lanthionine and β-methyllanthionine, which are introduced through enzyme-catalysed post-translational modification. Lantibiotics can be subdivided on the basis of the nature of the enzyme(s) which catalyse this reaction. Lantibiotic synthetases, generically designated LanM, which catalyse the dehydration of serines (and threonines) followed by the formation lanthionine (and β-methyllanthioine), are responsible for the synthesis of the largest subdivision, type 2. One can take advantage of the conserved nature of LanM proteins to screen for and bioinformatically characterize novel lantibiotic-encoding operons in genome-sequenced microorganisms. Having employed this strategy with success previously, here we update the investigation to reveal the existence of 124 LanM homologs encoded within genome-sequenced microbes. Further analysis focussed specifically on 9 novel lantibiotic gene clusters in Anaerocellum thermophilum DSM 6725, Anaerococcus tetradius ATCC 35098, Corynebacterium matruchotti ATCC 33806, Streptococcus suis 98HAH33, Geobacillus sp. G11MC16, Nostoc punctiforme PCC 73102 (× 2; one on plasmid and one on the chromosome) and Streptococcus pneumoniae CDC 0288-04 and TIGR4. Furthermore, screening of metagenomic datasets revealed 11 additional LanM-encoding genes from a variety of environments. The alignment of these LanM proteins facilitated a detailed investigation of conserved domains and led to the design of an improved set of degenerate primers which can be employed in the laboratory to identify strains containing type 2 lantibiotic gene clusters.     

Presence of GadD1 glutamate decarboxylase in selected Listeria monocytogenes strains is associated with an ability to grow at low pH

The glutamate decarboxylase (GAD) system is critical to the survival of Listeria monocytogenes LO28 at low-pH stress (相似文献   

Bacteriocins: developing innate immunity for food

Bacteriocins are bacterially produced antimicrobial peptides with narrow or broad host ranges. Many bacteriocins are produced by food-grade lactic acid bacteria, a phenomenon which offers food scientists the possibility of directing or preventing the development of specific bacterial species in food. This can be particularly useful in preservation or food safety applications, but also has implications for the development of desirable flora in fermented food. In this sense, bacteriocins can be used to confer a rudimentary form of innate immunity to foodstuffs, helping processors extend their control over the food flora long after manufacture.     

Structural characterization of lacticin 3147, a two-peptide lantibiotic with synergistic activity

Lantibiotics are antibacterial peptides isolated from bacterial sources that exhibit activity toward Gram-positive organisms and are usually several orders of magnitude more potent than traditional antibiotics such as penicillin. They contain a number of unique structural features including dehydro amino acid and lanthionine (thioether) residues. Introduced following ribosomal translation of the parent peptide, these moieties render conventional methods of peptide analysis ineffective. We report herein a new method using nickel boride (Ni(2)B), in the presence of deuterium gas, to reduce dehydro side chains and reductively desulfurize lanthionine bridges found in lantibiotics. Using this approach, it is possible to identify and distinguish the original locations of dehydro side chains and lanthionine bridges by traditional peptide sequencing (Edman degradation) followed by mass spectrometry. The strategy was initially verified using nisin A, a structurally well characterized lantibiotic, and subsequently extended to the novel two-component lantibiotic, lacticin 3147, produced by Lactococcus lactis subspecies lactis DPC3147. The primary structures of both lacticin 3147 peptides were then fully assigned by use of multidimensional NMR spectroscopy, showing that lacticin 3147 A1 has a specific lanthionine bridging pattern which resembles the globular type-B lantibiotic mersacidin, whereas the A2 peptide is a member of the elongated type-A lantibiotic class. Also obtained by NMR were solution conformations of both lacticin 3147 peptides, indicating that A1 may adopt a conformation similar to that of mersacidin and that the A2 peptide adopts alpha-helical structure. These results are the first of their kind for a synergistic lantibiotic pair (only four such pairs have been reported to date).