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N-(4-Bromobenzyl)-2-(5,6-dimethyl-1H-benzo[d]imid-azol-2-yl)benzeneamine
Get PDFN-(4-Bromobenzyl)-2-(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)benzeneamine was obtained by condensation of N-(4-bromobenzyl)-3,1-benzoxazine-2,4-dione (N-(4-bromobenzyl)isatoic anhydride) with 4,5-dimethyl-1,2-phenylenediamine in refluxing acetic acid. This is a rare example of condensation of N-substituted 3,1-benzoxazine-2,4-dione with 1,2-phenylenediamine, which resulted in the formation of a benzimidazole derivative with a moderate yield. Crystallographic studies and initial biological screening were performed for the obtained produc
A cryotechnique-based method for low abundance protein immunolocalization in tomato ( Solanum lycopersicum ) roots infected with a nematode, Globodera rostochiensis
Get PDFPseudomonas aeruginosa partitioning protein ParB acts as a nucleoid-associated protein binding to multiple copies of a parS-related motif
Get PDFParA and ParB homologs are involved in accurate chromosome segregation in bacteria. ParBs participate in the separation of ori domains by binding to parS palindromes, mainly localized close to oriC. In Pseudomonas aeruginosa neither ParB deficiency nor modification of all 10 parSs is lethal. However, such mutants show not only defects in chromosome segregation but also growth retardation and motility dysfunctions. Moreover, a lack of parB alters expression of over 1000 genes, suggesting that ParB could interact with the chromosome outside its canonical parS targets. Here, we show that indeed ParB binds specifically to hundreds of sites in the genome. ChIP-seq analysis revealed 420 ParB-associated regions in wild-type strain and around 1000 in a ParB-overproducing strain and in various parS mutants. The vast majority of the ParB-enriched loci contained a heptanucleotide motif corresponding to one arm of the parS palindrome. All previously postulated parSs, except parS5, interacted with ParB in vivo. Whereas the ParB binding to the four parS sites closest to oriC, parS1-4, is involved in chromosome segregation, its genome-wide interactions with hundreds of parS half-sites could affect chromosome topology, compaction and gene expression, thus allowing P. aeruginosa ParB to be classified as a nucleoid-associated protein
Activity and structure of EcoKMcrA.
Get PDFEscherichia coli McrA (EcoKMcrA) acts as a methylcytosine and hydroxymethylcytosine dependent restriction endonuclease. We present a biochemical characterization of EcoKMcrA that includes the first demonstration of its endonuclease activity, small angle X-ray scattering (SAXS) data, and a crystal structure of the enzyme in the absence of DNA. Our data indicate that EcoKMcrA dimerizes via the anticipated C-terminal HNH domains, which together form a single DNA binding site. The N-terminal domains are not homologous to SRA domains, do not interact with each other, and have separate DNA binding sites. Electrophoretic mobility shift assay (EMSA) and footprinting experiments suggest that the N-terminal domains can sense the presence and sequence context of modified cytosines. Pyrrolocytosine fluorescence data indicate no base flipping. In vitro, EcoKMcrA DNA endonuclease activity requires Mn2+ ions, is not strictly methyl dependent, and is not observed when active site variants of the enzyme are used. In cells, EcoKMcrA specifically restricts DNA that is modified in the correct sequence context. This activity is impaired by mutations of the nuclease active site, unless the enzyme is highly overexpressed
The Bacteroidetes Q-Rule: Pyroglutamate in Signal Peptidase I Substrates
Get PDFBacteroidetes feature prominently in the human microbiome, as major colonizers of the gut and clinically relevant pathogens elsewhere. Here, we reveal a new Bacteroidetes specific feature in the otherwise widely conserved Sec/SPI (Sec translocase/signal peptidase I) pathway. In Bacteroidetes, but not the entire FCB group or related phyla, signal peptide cleavage exposes N-terminal glutamine residues in most SPI substrates. Reanalysis of published mass spectrometry data for five Bacteroidetes species shows that the newly exposed glutamines are cyclized to pyroglutamate (also termed 5-oxoproline) residues. Using the dental pathogen Porphyromonas gingivalis as a model, we identify the PG2157 (also called PG_RS09565, Q7MT37) as the glutaminyl cyclase in this species, and map the catalytic activity to the periplasmic face of the inner membrane. Genetic manipulations that alter the glutamine residue immediately after the signal peptide in the pre-pro-forms of the gingipains affect the extracellular proteolytic activity of RgpA, but not RgpB and Kgp. Glutamine statistics, mass spectrometry data and the mutagenesis results show that the N-terminal glutamine residues or their pyroglutamate cyclization products do not act as generic sorting signals
2′-Deoxy-2′-azidonucleoside analogs: synthesis and evaluation of antitumor and antimicrobial activity
Get PDFA series of ten pyrimidine nucleosides modified in 2′ position with azide or amine group was tested for the antibacterial, antifungal and cytotoxic activity. The cytotoxic effect was determined on three cancer (CCRF-CEM, MCF7, HeLa) and one normal (HEK293) cell lines, while antibacterial activity was evaluated on five bacterial strains. Among others, 2′-azido-2′deoxycytidine and 2′-amino-2′-deoxycytidine exhibited the strongest antiproliferative activity at 200 μM concentration, decreasing the viability of CCRF-CEM cells to 33 ± 1 and 36 ± 2%, respectively. Newly synthesized 2′-amino-2′-deoxythymidine exhibited cytotoxic effect exclusively toward HeLa cancer cell line, but not toward the normal HEK293 cells. Also, investigated compounds did not exhibit any antibacterial or antifungal activity at a concentration of 40 mM. The obtained results suggest that the presence of cytosine base is desirable for the appearance of cytotoxic effect, while the structural variations of the sugar ring play a minor role. Future modification of 2′-amino-2′-deoxythymidine could be a promising way to obtain more active anticancer substances
Characteristics of the conjugative transfer system of the IncM plasmid pCTX-M3 and identification of its putative regulators
Get PDFPlasmid conjugative transfer systems comprise type IV secretion systems (T4SS) coupled to DNA processing and replication. The T4SSs are divided into two phylogenetic subfamilies, namely, IVA and IVB or based on the phylogeny of the VirB4 ATPase into eight groups. The conjugation system of the IncM group plasmid pCTX-M3, from Citrobacter freundii, is classified in the IVB subfamily and in the MPFI group, as are the conjugation systems of IncI1 group plasmids. Although the majority of the conjugative genes of the IncM and IncI1 plasmids display conserved synteny, there are several differences. Here, we present a deletion analysis of 27 genes in the conjugative transfer regions of pCTX-M3. Notably, the deletion of either of two genes dispensable for conjugative transfer, namely, orf35 and orf36, resulted in an increased plasmid mobilization efficiency. Transcriptional analysis of the orf35 and orf36 deletion mutants suggested an involvement of these genes in regulating the expression of conjugative transfer genes. We also revised the host range of the pCTX-M3 replicon by finding that this replicon is unable to support replication in Agrobacterium tumefaciens, Ralstonia eutropha and Pseudomonas putida although its conjugation system is capable of introducing plasmids bearing oriTpCTX-M3 into these bacteria, which are representatives of Alpha-, Beta- and Gammaproteobacteria, respectively. Thus, the conjugative transfer system of pCTX-M3 has a much broader host range than does its replicon.
Importance
Horizontal gene transfer is responsible for rapid changes in bacterial genomes, and the conjugative transfer of plasmids has a great impact on the plasticity of bacteria. Here, we present a deletion analysis of the conjugative transfer system genes of the pCTX-M3 plasmid of the IncM group, which is responsible for the dissemination of antibiotic resistance genes in Enterobacteriaceae. We found that deletion of the orf35 or orf36 genes, which are dispensable for conjugative transfer, increased the plasmid mobilization efficiency. RT-qPCR analysis suggested the involvement of orf35 and orf36 in regulating the expression of transfer genes. We also revised the host range of pCTX-M3 by showing that its conjugative transfer system has a much broader host range than does its replicon
Meconium proteins as a source of biomarkers for the assessment of the intrauterine environment of the fetus
Get PDFIntrauterine environmental factors can be associated with perinatal complications and long-term health outcomes although the underlying mechanisms remain poorly defined. Meconium formed exclusively in utero and passed naturally by a neonate may contain proteins which characterise the intrauterine environment. The aim of the study was proteomic analysis of the composition of meconium proteins and their classification by biological function. Proteomic techniques combining isoelectrofocussing fractionation and LC-MS/MS analysis were used to study the protein composition of a meconium sample obtained by pooling 50 serial meconium portions from 10 healthy full-term neonates. The proteins were classified by function based on the literature search for each protein in the PubMed database. A total of 946 proteins were identified in the meconium, including 430 proteins represented by two or more peptides. When the proteins were classified by their biological function the following were identified: immunoglobulin fragments and enzymatic, neutrophil-derived, structural and fetal intestine-specific proteins. Meconium is a rich source of proteins deposited in the fetal intestine during its development in utero. A better understanding of their specific biological functions in the intrauterine environment may help to identify these proteins which may serve as biomarkers associated with specific clinical conditions/diseases with the possible impact on the fetal development and further health consequences in infants, older children and adults
Time-Course Microarray Analysis Reveals Differences between Transcriptional Changes in Tomato Leaves Triggered by Mild and Severe Variants of Potato Spindle Tuber Viroid
Get PDFViroids are small non-capsidated non-coding RNA replicons that utilize host factors for efficient propagation and spread through the entire plant. They can incite specific disease symptoms in susceptible plants. To better understand viroid-plant interactions, we employed microarray analysis to observe the changes of gene expression in ‘Rutgers’ tomato leaves in response to the mild (M) and severe (S23) variants of potato spindle tuber viroid (PSTVd). The changes were analyzed over a time course of viroid infection development: (i) the pre-symptomatic stage, (ii) early symptoms, (iii) full spectrum of symptoms and (iv) the so-called ‘recovery’ stage, when stem regrowth was observed in severely affected plants. Gene expression profiles differed depending on stage of infection and variant. In S23-infected plants, the expression of over 3,000 genes was affected, while M-infected plants showed 3-fold fewer differentially expressed genes, only 20% of which were specific to the M variant. The differentially expressed genes included many genes related to stress; defense; hormone metabolism and signaling; photosynthesis and chloroplasts; cell wall; RNA regulation, processing and binding; protein metabolism and modification and others. The expression levels of several genes were confirmed by nCounter analysis
ERCC1-deficient cells and mice are hypersensitive to lipid peroxidation
Get PDFLipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1-/- mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl4 and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1-/- cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1-/- cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats