217 research outputs found
7,8-Dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases
Hydroxyl radicals predominantly react with the C(8) of purines forming 7,8-dihydro-8-oxoguanine (8oxoG) and 7,8-dihydro-8-oxoadenine (8oxoA) adducts, which are highly mutagenic in mammalian cells. The majority of oxidized DNA bases are removed by DNA glycosylases in the base excision repair pathway. Here, we report for the first time that human thymine-DNA glycosylase (hTDG) and Escherichia coli mismatch-specific uracil-DNA glycosylase (MUG) can remove 8oxoA from 8oxoA*T, 8oxoA*G and 8oxoA*C pairs. Comparison of the kinetic parameters of the reaction indicates that full-length hTDG excises 8oxoA, 3,N(4)-ethenocytosine (epsilonC) and T with similar efficiency (k(max) = 0.35, 0.36 and 0.16 min(-1), respectively) and is more proficient as compared with its bacterial homologue MUG. The N-terminal domain of the hTDG protein is essential for 8oxoA-DNA glycosylase activity, but not for epsilonC repair. Interestingly, the TDG status had little or no effect on the proliferation rate of mouse embryonic fibroblasts after exposure to gamma-irradiation. Nevertheless, using whole cell-free extracts from the DNA glycosylase-deficient murine embryonic fibroblasts and E. coli, we demonstrate that the excision of 8oxoA from 8oxoA*T and 8oxoA*G has an absolute requirement for TDG and MUG, respectively. The data establish that MUG and TDG can counteract the genotoxic effects of 8oxoA residues in vivo
Aberrant repair initiated by mismatch-specific thymine-DNA glycosylases provides a mechanism for the mutational bias observed in CpG islands
The human thymine-DNA glycosylase (TDG) initiates
the base excision repair (BER) pathway to remove
spontaneous and induced DNA base damage. It was
first biochemically characterized for its ability to remove
T mispaired with G in CpG context. TDG is
involved in the epigenetic regulation of gene expressions
by protecting CpG-rich promoters from de
novo DNA methylation. Here we demonstrate that
TDG initiates aberrant repair by excising T when it
is paired with a damaged adenine residue in DNA
duplex. TDG targets the non-damaged DNA strand
and efficiently excises T opposite of hypoxanthine
(Hx), 1,N6-ethenoadenine, 7,8-dihydro-8-oxoadenine
and abasic site in TpG/CpX context, where X is
a modified residue. In vitro reconstitution of BER
with duplex DNA containing Hx•T pair and TDG results
in incorporation of cytosine across Hx. Furthermore,
analysis of the mutation spectra inferred from
single nucleotide polymorphisms in human population
revealed a highly biased mutation pattern within
CpG islands (CGIs), with enhanced mutation rate at
CpA and TpG sites. These findings demonstrate that
under experimental conditions used TDG catalyzes
sequence context-dependent aberrant removal of
thymine, which results in TpG, CpA→CpGmutations,
thus providing a plausible mechanism for the putative
evolutionary origin of the CGIs in mammalian
genomes
Autophagy in dental tissues: a double-edged sword
Biotechnology and Biological Sciences Research Council [BB/L02392X/1]SCI(E)PubMedEDITORIAL [email protected]
Frizzled-1 receptor regulates adult hippocampal neurogenesis
Indexación: Web of Science; Scopus.Background: In the adult hippocampus new neurons are continuously generated from neural stem cells (NSCs) present at the subgranular zone of the dentate gyrus. This process is controlled by Wnt signaling, which plays a complex role in regulating multiple steps of neurogenesis including maintenance, proliferation and differentiation of progenitor cells and the development of newborn neurons. Differential effects of Wnt signaling during progression of neurogenesis could be mediated by cell-type specific expression of Wnt receptors. Here we studied the potential role of Frizzled-1 (FZD1) receptor in adult hippocampal neurogenesis.
Results: In the adult dentate gyrus, we determined that FZD1 is highly expressed in NSCs, neural progenitors and immature neurons. Accordingly, FZD1 is expressed in cultured adult hippocampal progenitors isolated from mouse brain. To evaluate the role of this receptor in vivo we targeted FZD1 in newborn cells using retroviral-mediated RNA interference. FZD1 knockdown resulted in a marked decrease in the differentiation of newborn cells into neurons and increased the generation of astrocytes, suggesting a regulatory role for the receptor in cell fate commitment. In addition, FZD1 knockdown induced an extended migration of adult-born neurons within the granule cell layer. However, no differences were observed in total dendritic length and dendritic arbor complexity between control and FZD1-deficient newborn neurons.
Conclusions: Our results show that FZD1 regulates specific stages of adult hippocampal neurogenesis, being required for neuronal differentiation and positioning of newborn neurons into the granule cell layer, but not for morphological development of adult-born granule neurons.https://molecularbrain.biomedcentral.com/articles/10.1186/s13041-016-0209-
A framework to develop semiautomated surveillance of surgical site infections: An international multicenter study
Objective: Automated surveillance of healthcare-associated infections reduces workload and improves standardization, but it has not yet been adopted widely. In this study, we assessed the performance and feasibility of an easy implementable framework to develop algorithms for semiautomated surveillance of deep incisional and organ-space surgical site infections (SSIs) after orthopedic, cardiac, and colon surgeries. Design: Retrospective cohort study in multiple countries. Methods: European hospitals were recruited and selected based on the availability of manual SSI surveillance data from 2012 onward (reference standard) and on the ability to extract relevant data from electronic health records. A questionnaire on local manual surveillance and clinical practices was administered to participating hospitals, and the information collected was used to pre-emptively design semiautomated surveillance algorithms standardized for multiple hospitals and for center-specific application. Algorithm sensitivity, positive predictive value, and reduction of manual charts requiring review were calculated. Reasons for misclassification were explored using discrepancy analyses. Results: The study included 3 hospitals, in the Netherlands, France, and Spain. Classification algorithms were developed to indicate procedures with a high probability of SSI. Components concerned microbiology, prolonged length of stay or readmission, and reinterventions. Antibiotics and radiology ordering were optional. In total, 4,770 orthopedic procedures, 5,047 cardiac procedures, and 3,906 colon procedures were analyzed. Across hospitals, standardized algorithm sensitivity ranged between 82% and 100% for orthopedic surgery, between 67% and 100% for cardiac surgery, and between 84% and 100% for colon surgery, with 72%-98% workload reduction. Center-specific algorithms had lower sensitivity. Conclusions: Using this framework, algorithms for semiautomated surveillance of SSI can be successfully developed. The high performance of standardized algorithms holds promise for large-scale standardization
The human DNA glycosylases NEIL1 and NEIL3 excise psoralen-induced DNA-DNA cross-links in a four-stranded DNA structure
Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and
human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus
egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing
either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork
bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks
GABAB Receptor Subunit GB1 at the Cell Surface Independently Activates ERK1/2 through IGF-1R Transactivation
BACKGROUND: Functional GABA(B) receptor is believed to require hetero-dimerization between GABA(B1) (GB1) and GABA(B2) (GB2) subunits. The GB1 extracellular domain is required for ligand binding, and the GB2 trans-membrane domain is responsible for coupling to G proteins. Atypical GABA(B) receptor responses observed in GB2-deficient mice suggested that GB1 may have activity in the absence of GB2. However the underlying mechanisms remain poorly characterized. METHODOLOGY/PRINCIPAL FINDINGS: Here, by using cells overexpressing a GB1 mutant (GB1asa) with the ability to translocate to the cell surface in the absence of GB2, we show that GABA(B) receptor agonists, such as GABA and Baclofen, can induce ERK1/2 phosphorylation in the absence of GB2. Furthermore, we demonstrate that GB1asa induces ERK1/2 phosphorylation through Gi/o proteins and PLC dependent IGF-1R transactivation. CONCLUSIONS/SIGNIFICANCE: Our data suggest that GB1 may form a functional receptor at the cell surface in the absence of GB2
Functioning of the dimeric GABA(B) receptor extracellular domain revealed by glycan wedge scanning
The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA
is made up of two subunits, GABA(B1) and GABA(B2). GABA(B1) binds agonists,
whereas GABA(B2) is required for trafficking GABA(B1) to the cell surface,
increasing agonist affinity to GABA(B1), and activating associated G proteins.
These subunits each comprise two domains, a Venus flytrap domain (VFT) and a
heptahelical transmembrane domain (7TM). How agonist binding to the GABA(B1)
VFT leads to GABA(B2) 7TM activation remains unknown. Here, we used a glycan
wedge scanning approach to investigate how the GABA(B) VFT dimer controls
receptor activity. We first identified the dimerization interface using a
bioinformatics approach and then showed that introducing an N-glycan at this
interface prevents the association of the two subunits and abolishes all
activities of GABA(B2), including agonist activation of the G protein. We also
identified a second region in the VFT where insertion of an N-glycan does not
prevent dimerization, but blocks agonist activation of the receptor. These data
provide new insight into the function of this prototypical GPCR and demonstrate
that a change in the dimerization interface is required for receptor
activation
Specificity of Transmembrane Protein Palmitoylation in Yeast
Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs), characterized by the presence of a conserved 50- aminoacids domain called “Asp-His-His-Cys- Cysteine Rich Domain” (DHHC-CRD). There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether
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