Weekend deck Flashcards
what is the redox potential of guanine
guanine 1.29, adenine 1.42, cytosine 1.6 and thymine 1.7.
caused by the unsaturated N7–C8 bond
What time point does GD 17 correspond to
- The degree of brain development on GD 17 in mice corresponds approximately to that in weeks 12–13 in human fetuses (Clancy et al., 2007);
Why do you think there are such high rates of INCREASe in gene expression
Recent reports include evidence that in mice ethanol consumption leads to rapid increase in histone acetylation in the brain with the acetylmoiety arising from ethanol [74]. This acetylation is reported to alter gene expression in hippocampus and to affect cellular mechanisms related to learning.
How do CYPS generate ROS
○ CYP2E1 generates reactive oxygen species (ROS) during the metabolism of certain substrates like ethanol, during the oxidation process, it can “leak” electrons to oxygen molecules, leading to the production of superoxide radicals and other ROS, particularly when the metabolic process is not fully coupled
Specifically, during process, the first electron is passed to the hene of CYP2E1 and oxygen is bound, this generates superoxide bound to the heme of CYP2E1. Occasionally the superoxide will break down –> releasing superoxide. This is called the uncoupling reaction because the oxuge does not end up on the substrate
INihibitors of OGG1
environmental inhibitor is cadmium
small drug molecule TH5487
Catalytic mechanism of OGG1
lycosylase catalytic activity involves two amino acids -> Lysine 249 and Asp268
○ Lys249 attacks the glycosidic bond of 8-oxoG to release the residue, C-1’ of the deoxyribose sugar, formation of a covalently linked enzyme-substrate intermediate, and rearranges into a Schiff base, eventually undergoes a lyase reaction to expel the 8-oxoG base via beta-elimination and leaving a nick in the DNA backbone. Rate limiting step of this reaction is the formation of the Schiff base -> opening of the ribose ring within the Lyse249-ribose covalent adduct
○ Asp268 role is still unclear but may donate a proton -> protonation of ε-amino group of Lys249 to help stabilize the negative charge on the 8-xooG (Schyman et al 2005; Kungland et al 2007), Asp 268 may also on the deoxyribose sugar as a charge-compensating residue. Mutation (to asparagine or glutamine or glutamic acid results in binding of DNA but little enzymatic activity (Normal et al 2003; Severa et al 2007)
Gly42 may also be responsible for the recognition of the and deposition of the 8-oxoG in the catalytic core -> via a hydrogen bond (crenshaw et al 2012)
Ogg1 protien interactions
DNA repair: APE1, XRCC1
Transcription facotrs: NF-kB, SP1, CUX1/2
Chromatin: cohesion, SATB1
what is developing at GD 17
GD 17
round the timing of gestational exposure to EtOH (GD 17), the lobules of the cerebellum are forming, a brain region implicated in motor control (CHEN et al. 2017). Thus, different behavioural effects may be observed, depending on the timing of exposure. Around the timing of exposure used herein, lamination of the neocortex is also forming (CHEN et al. 2017), which has been implicated in integrating and recalling memories (PRESTON AND EICHENBAUM2013).
Furthermore, GD 17 immediately predates the sensitive time window for NF-kB signaling in neurite outgrowth (embryonic day 18 to postnatal day 1) (GUTIERREZ et al. 2005), and synaptogenesis which occurs shortly after birth (postnatal day 3) (CHEN et al. 2017).
How does H2O2 modify proteins
- Selectively oxidize sulfhydryl groups of specific cysteine (cys) residues, acting as a sulfur switch.
* At physiologically relevant concentrations of H2O2 the oxidative modification are reversible, but higher exposures lead to excessive and irreversible s-oxidation (sulfinic acid) –> loss of protein
○ Sulfur-containing thiol groups (R-SH) can exist in multiple oxidation states, ROS oxidation to sulfenic acid (RSOH) intermediates which can react with neighboring thiol groups to form intra/inter molecular protein-protein disulfides, wit with GSH to produce GSH-protein disulfide
* Regulate:
○ protein tyrosine phosphatases
○ Protein tyrosine kinases
○ Small G proteins
○ Transcription factors
▪ Nrf2
▪ ASK-1
▪ NF-kB
Function of PBN
ee radical spin trapping agent PBN
§ Contain a nitroso group that can scavenge ROS
§ Block inflammatory pathways, can inhibit:
(1) the induction iNOS, thereby decreasing NO* production
(2) activation of NFκB, which is involved in ROS signaling;
(3) inducible cyclooxygenase (COX) 2 mRNA expression and COX catalytic activity (Kotake et al., 1998), involved in xenobiotic bioactivation to free radical intermediates;
(4) transcription of proinflammatory cytokines such as tumor necrosis factor-α (TNF- α) and interferon-gamma (IFN-γ), which is correlated with decreased activation of the NF-κB and activator protein-1 (AP-1) transcription factors (Sang et al., 1999);
(5) cleave of caspase-3, which is involved in apoptosis (McLaughlin et al., 2003); and,
(6) activation of NOXs (Chang et al., 2009) which are activated in the embryo by in utero EtOH exposure, increasing ROS production, embryonic DNA oxidation and structural teratogenesis (Dong et al., 2010)
Types of pathway anlsysi
Functional pathway analysis
over representation analysis
functional class sorting
pathway topology
miRNA function for silencing
a. microRNAs (miRNAs) are small, non-coding RNAs (~22 nucleotides) that regulate gene expression post-transcriptionally by either inhibiting translation or promoting mRNA degradation.
i. transcribed as primary miRNAs (pri-miRNAs) and processed in the nucleus by the Drosha-DGCR8 complex into precursor miRNAs (pre-miRNAs)
ii. exported to the cytoplasm via Exportin-5.
iii. Dicer cleaves the pre-miRNA into a mature miRNA duplex, and one strand (the guide strand) is incorporated into the RNA-induced silencing complex (RISC), primarily interacting with Argonaute (AGO) proteins.
iv. miRNAs bind to target mRNAs, typically in the 3′ untranslated region (3′ UTR), leading to either translational repression (if binding is partial) or mRNA degradation (if binding is near-perfect).
How does GC-MS/MS with flame ionizng detections work?
Headspace Sampling (HS) Analyzes the volatile fraction of a sample without injecting the sample itself, volatile components partition into the gas phase.
Gas Chromatography (GC) Separates volatile components based on their boiling points and polarity.
Mass Spectrometry (MS) Identifies and quantifies compounds based on their mass-to-charge ratio (m/z), the analyte molecules are ionized, fragmented, and detected by the mass spectrometer. Provides a unique mass spectrum for each compound, aiding in identification.
D.Detection with FID, The eluted ethanol enters the FID detector, where it is burned in a hydrogen-air flame. The combustion ionizes ethanol molecules, generating a measurable electrical current. The signal intensity is proportional to the ethanol concentration in the sample.
Define NDDs
○ Neurodevelopmental disorders (NDDs) define a range of conditions that involve a disruption in brain development leading to altered function or behaviour5. This change in brain function or behaviour can manifest in difficulties in various domains, such as cognitive, social, emotional, or physical abilities
What is the data behind OGG1 interactrions with NF-KB and SP1?
NF-kB inhibition with antisense oligonucleotides prevented ROS-initiated teratogenesis by phenytoin (Kennedy et al. 2004)*
* ChIP studies à OGG1 and NF-kB colocalize at promoter regions upon TNF-alpha exposure (Pan et al. 2004; Hai et al. 2018)
* Reporter gene (Cxcl2) 8-oxoG accumulation and OGG1 binding was essential for NF-kB mediated transcription (Pan et al. 2017)
* OGG1 knockdow à altered allergic immune response in mouse airway epithelium (Bacsi et al. 2013)
* OGG1 inhibition supresses proinflammatory gene expression and inflammation (Visnes et al. 2018)
Pull down assays à RelA and OGG1 interaction (Ba et al. 2014)
olive tail moment
percent tail DNA x distance between the means of the head and tail distributions
sex differentiation in mice
the differentiation of gonads (testes or ovaries), which will produce sex hormones, happens at embryonic day (E) 10.5 to 11 in mice. This process is driven by the Sry gene (Sex-determining region Y) located on the Y chromosome:
The gonads are fully differentiated into either testes or ovaries by GD 17. In males (XY), the testes have developed with Sertoli cells supporting sperm formation, while in females (XX), the ovaries are developing with oocyte formation.
what is LTP
Long-term potentiation (LTP) is a persistent strengthening of synaptic connections following repeated stimulation, which enhances synaptic transmission and is considered a key mechanism underlying learning and memory. It involves increased postsynaptic receptor activity, primarily through NMDA and AMPA receptors, leading to calcium influx, activation of signaling pathways (e.g., CaMKII, PKA, MAPK). Synaptic activity depolarizes the postsynaptic neuron, NMDA receptors open, allowing Ca²⁺ to enter the cell. The influx of Ca²⁺ binds to calmodulin, activating CaMKII, which then phosphorylates AMPA receptors, increasing their conductance and promoting their insertion into the synaptic membrane, leading to enhanced synaptic transmission.
○ LTP can be classified into early-phase and late-phase LTP, The late-phase LTP is thought to contribute to memory maintenance
- What time point does GD 17 correspond to
- The degree of brain development on GD 17 in mice corresponds approximately to that in weeks 12–13 in human fetuses (Clancy et al., 2007);
Epigenetic markers
DNA methylation is generally associated with gene silencing
Acetylation of lysine residues (e.g., H3K9ac, H3K27ac) is typically associated with gene activation.
Methylation of lysine residues (e.g., H3K4me3, H3K27me3) can be associated with either gene activation or repression, depending on the specific modification and context.
MIr-9 pathway targets
miR-9, a microRNA, targets various pathways, including the Notch signaling pathway, PI3K/Akt/autophagy pathway, and the HIF-1α/VEGF signaling pathway, and also plays a role in microglial activation and axonal extension.
esterus cycle effect
The estrus cycle in mice, consisting of proestrus, estrus, metestrus, and diestrus, influences learning and memory, with some studies showing that estrogen-dominant stages (proestrus, estrus) can worsen cognitive function, while progesterone-dominant stages (metestrus, diestrus) may have a protective effect.
