Construction of targeted and integrative promoter-reporter plasmids pDK-K and pDK-G to measure gene expression activity in Haemophilus parasuis

2019 ◽  
Vol 134 ◽  
pp. 103565 ◽  
Author(s):  
Ke Dai ◽  
Zhen Yang ◽  
Yung-Fu Chang ◽  
Lvqin He ◽  
Sanjie Cao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Haemophilus Parasuis ◽  
Expression Activity ◽  
Measure Gene Expression

scholarly journals The Effect of Sulforaphane on Glyoxalase I Expression and Activity in Peripheral Blood Mononuclear Cells

Nutrients ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 1773 ◽  
Author(s):  
Michela Alfarano ◽  
Donato Pastore ◽  
Vincenzo Fogliano ◽  
Casper Schalkwijk ◽  
Teresa Oliviero
Keyword(s):  
Gene Expression ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Limit Of Detection ◽  
Peripheral Blood Mononuclear ◽  
Potential Health ◽  
Age Related ◽  
Blood Mononuclear Cells ◽  
Expression Activity

Studies demonstrate that the potential health-beneficial effect of sulforaphane (SR), a compound formed in broccoli, is the result of a number of mechanisms including upregulation of phase two detoxification enzymes. Recent studies suggest that SR increases expression/activity of glyoxalase 1 (Glo1), an enzyme involved in the degradation of methylglyoxal, is major precursor of advanced glycation end products. Those compounds are associated with diabetes complications and other age-related diseases. In this study, the effect of SR on the expression/activity of Glo1 in peripheral blood mononuclear cells (PBMCs) from 8 healthy volunteers was investigated. PBMCs were isolated and incubated with SR (2.5 μM-concentration achievable by consuming a broccoli portion) for 24 h and 48 h. Glo1 activity/expression, reduced glutathione (GSH), and glutathione-S-transferase gene expression were measured. Glo1 activity was not affected while after 48 h a slight but significant increase of its gene expression (1.03-fold) was observed. GSTP1 expression slightly increased after 24 h incubation (1.08-fold) while the expressions of isoform GSTT2 and GSTM2 were below the limit of detection. GSH sharply decreased, suggesting the formation of GSH-SR adducts that may have an impact SR availability. Those results suggest that a regular exposure to SR by broccoli consumption or SR supplements may enhance Glo1.

scholarly journals NPY and sbGnRH gene expression in juvenile and adult male Brazilian flounder Paralichthys orbignyanus

2011 ◽  
Vol 41 (11) ◽  
pp. 1927-1930 ◽  
Author(s):  
Vinicius Farias Campos ◽  
Tiago Veiras Collares ◽  
Fabiana Kömmling Seixas ◽  
João Carlos Deschamps ◽  
Luis Fernando Fernandes Marins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Extraction ◽  
Sea Bream ◽  
Mrna Levels ◽  
Adult Males ◽  
Adult Fish ◽  
Cdna Synthesis ◽  
Hypothalamic Regulation ◽  
Measure Gene Expression ◽  
Paralichthys Orbignyanus

The objective of this study was to evaluate neuropeptide Y (NPY) and sea bream gonadotropin-release hormone (sbGnRH) gene expression in juvenile and adult males of Brazilian flounder. Hypothalamuses from fish were sampled for total RNA extraction. After cDNA synthesis, real-time PCR was used to measure gene expression. NPY showed approximately 2-fold increases in their mRNA levels while sbGnRH showed 3-fold increases in adult fish. These results suggest that these peptides could be involved on hypothalamic regulation of Brazilian flounder sexual maturation.

scholarly journals The effect of hairpin loop on the structure and gene expression activity of the long-loop G-quadruplex

2021 ◽  
Author(s):  
Subramaniyam Ravichandran ◽  
Maria Razzaq ◽  
Nazia Parveen ◽  
Ambarnil Ghosh ◽  
Kyeong Kyu Kim
Keyword(s):  
Gene Expression ◽  
Rna Structure ◽  
Biological Significance ◽  
Cellular Functions ◽  
Hairpin Loops ◽  
G Quadruplex ◽  
Reporter Assays ◽  
Expression Activity ◽  
The Stability ◽  
And Function

Abstract G-quadruplex (G4), a four-stranded DNA or RNA structure containing stacks of guanine tetrads, plays regulatory roles in many cellular functions. So far, conventional G4s containing loops of 1–7 nucleotides have been widely studied. Increasing experimental evidence suggests that unconventional G4s, such as G4s containing long loops (long-loop G4s), play a regulatory role in the genome by forming a stable structure. Other secondary structures such as hairpins in the loop might thus contribute to the stability of long-loop G4s. Therefore, investigation of the effect of the hairpin-loops on the structure and function of G4s is required. In this study, we performed a systematic biochemical investigation of model G4s containing long loops with various sizes and structures. We found that the long-loop G4s are less stable than conventional G4s, but their stability increased when the loop forms a hairpin (hairpin-G4). We also verified the biological significance of hairpin-G4s by showing that hairpin-G4s present in the genome also form stable G4s and regulate gene expression as confirmed by in cellulo reporter assays. This study contributes to expanding the scope and diversity of G4s, thus facilitating future studies on the role of G4s in the human genome.

scholarly journals Brain gene expression in a novel mouse model of postpartum mood disorder

2019 ◽  
Vol 10 (1) ◽  
pp. 168-174
Author(s):  
Trevor Humby ◽  
William Davies
Keyword(s):  
Gene Expression ◽  
Pathway Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Late Pregnancy ◽  
Maternal Behaviour ◽  
Aberrant Expression ◽  
Brain Gene Expression ◽  
False Discovery ◽  
Expression Activity ◽  
Polymerase Chain

Abstract Background Steroid sulfatase (STS) cleaves sulfate groups from steroid hormones; its expression/activity increases in late pregnancy and into the postpartum period. STS-deficient human and mouse mothers display elevated psychopathology and abnormal behaviour respectively; in mice, these effects can be partially normalised by antipsychotic (ziprasidone) administration. Methodology We compared brain gene expression in new mouse mothers administered the STS inhibitor 667-Coumate, or vehicle; significant changes were followed-up with pathway analysis and quantitative polymerase chain reaction (qPCR). Finally, the effects of combined 667-Coumate and ziprasidone administration on expression of the most robustly differentially-expressed genes were examined. Results Surprisingly, no between-group gene expression changes were detected at a False Discovery Rate (FDR)-corrected p<0.1. 1,081 unique expression changes were detected at p<0.05, two top hits were verified by qPCR, and pathway analysis indicated enrichment of genes involved in olfactory transduction. The expression of Stoml3 and Cyp2g1 was unaffected by ziprasidone administration. Conclusions Postpartum behavioural abnormalities in STS-deficient mothers are likely to be the culmination of many small gene expression changes. Our data are consistent with the idea that olfactory function is key to maternal behaviour in mice, and suggest that aberrant expression of olfactory system genes may underlie abnormal maternal behaviour in STS-deficient women.

scholarly journals Artificial Light at Night Influences Clock-Gene Expression, Activity, and Fecundity in the Mosquito Culex pipiens f. molestus

2019 ◽  
Vol 11 (22) ◽  
pp. 6220 ◽  
Author(s):  
Honnen ◽  
Kypke ◽  
Hölker ◽  
Monaghan
Keyword(s):  
Gene Expression ◽  
Culex Pipiens ◽  
Clock Gene ◽  
Clock Cycle ◽  
Diel Activity ◽  
Artificial Light ◽  
Light At Night ◽  
Clock Gene Expression ◽  
Expression Activity ◽  
And Behavior

Light is an important environmental cue, and exposure to artificial light at night (ALAN) may disrupt organismal physiology and behavior. We investigated whether ALAN led to changes in clock-gene expression, diel activity patterns, and fecundity in laboratory populations of the mosquito Culex pipiens f. molestus (Diptera, Culicidae), a species that occurs in urban areas and is thus regularly exposed to ALAN. Populations were kept under 16hours (h):8h light:dark cycles or were subjected to an additional 3.5 h of light (100–300 lx) in the evenings. ALAN induced significant changes in expression in all genes studied, either alone (period) or as an interaction with time (timeless, cryptochrome2, Clock, cycle). Changes were sex-specific: period was down-regulated in both sexes, cycle was up-regulated in females, and Clock was down-regulated in males. ALAN-exposed mosquitoes were less active during the extra-light phase, but exposed females were more active later in the night. ALAN-exposed females also produced smaller and fewer eggs. Our findings indicate a sex-specific impact of ALAN on the physiology and behavior of Culex pipiens f. molestus and that changes in clock-gene expression, activity, and fecundity may be linked.

scholarly journals A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System forClostridiumOrganisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)

2019 ◽  
Vol 85 (14) ◽  
Author(s):  
Hannah E. Streett ◽  
Katie M. Kalis ◽  
Eleftherios T. Papoutsakis
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Real Time ◽  
Protein Localization ◽  
Anaerobic Conditions ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Expression Activity ◽  
Microplate Reader

ABSTRACTVisualizing protein localization and characterizing gene expression activity in liveClostridiumcells is limited for lack of a real-time, highly fluorescent, oxygen-independent reporter system. Enzymatic reporter systems have been used successfully for many years withClostridiumspp.; however, these assays do not allow for real-time analysis of gene expression activity with flow cytometry or for visualizing protein localization through fusion proteins. Commonly used fluorescent reporter proteins require oxygen for chromophore maturation and cannot be used for most strictly anaerobicClostridiumorganisms. Here we show that the fluorescence-activating and absorption-shifting tag protein (FAST), when associated with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR; now commercially available) and other commercially available ligands, is highly fluorescent inClostridium acetobutylicumunder anaerobic conditions. Using flow cytometry and a fluorescence microplate reader, we demonstrated FAST as a reporter system by employing the promoters of theC. acetobutylicumthiolase (thl), acetoacetate decarboxylase (adc), and phosphotransbutyrylase (ptb) metabolic genes, as well as a mutant Pthland modified ribosome binding site (RBS) versions of Padcand Pptb. Flow cytometry-based sorting was efficient and fast in sorting FAST-expressing cells, and positively and negatively sorted cells could be effectively recultured. FAST was also used to tag and examine protein localization of the predicted cell division FtsZ partner protein, ZapA, to visualize the divisome localization in liveC. acetobutylicumcells. Our findings suggest that FAST can be used to further investigateClostridiumdivisomes and more broadly the localization and expression levels of other proteins inClostridiumorganisms, thus enabling cell biology studies with these organisms.IMPORTANCEFAST in association with the fluorogenic ligand HMBR is characterized as a successful, highly fluorescent reporter system inC. acetobutylicum. FAST can be used to distinguish between promoters in live cells using flow cytometry or a fluorescence microplate reader and can be used to tag and examine protein localization in live, anaerobically grown cells. Given that FAST is highly fluorescent under anaerobic conditions, it can be used in several applications of this and likely manyClostridiumorganisms and other strict anaerobes, including studies involving cell sorting, sporulation dynamics, and population characterization in pure as well as mixed cultures, such as those in various native or synthetic microbiomes and syntrophic cultures.

scholarly journals Systemically Administered Ligands of Toll-Like Receptor 2, -4, and -9 Induce Distinct Inflammatory Responses in the Murine Lung

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
H. Ehrentraut ◽  
R. Meyer ◽  
M. Schwederski ◽  
S. Ehrentraut ◽  
M. Velten ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inflammatory Responses ◽  
Pulmonary Inflammation ◽  
Myeloperoxidase Activity ◽  
Cpg Odn ◽  
Toll Like Receptor ◽  
Activity Assay ◽  
Murine Lung ◽  
Expression Activity ◽  
Toll Like Receptor 2

Objective. To determine whether systemically administered TLR ligands differentially modulate pulmonary inflammation.Methods. Equipotent doses of LPS (20 mg/kg), CpG-ODN (1668-thioat 1 nmol/g), or LTA (15 mg/kg) were determined via TNF activity assay. C57BL/6 mice were challenged intraperitoneally. Pulmonary NFκB activation (2 h) and gene expression/activity of key inflammatory mediators (4 h) were monitored.Results. All TLR ligands induced NFκB. LPS increased the expression of TLR2, 6, and the cytokines IL-1αβ, TNF-α, IL-6, and IL-12p35/p40, CpG-ODN raised TLR6, TNF-α, and IL12p40. LTA had no effect. Additionally, LPS increased the chemokines MIP-1α/β, MIP-2, TCA-3, eotaxin, and IP-10, while CpG-ODN and LTA did not. Myeloperoxidase activity was highest after LPS stimulation. MMP1, 3, 8, and 9 were upregulated by LPS, MMP2, 8 by CpG-ODN and MMP2 and 9 by LTA. TIMPs were induced only by LPS. MMP-2/-9 induction correlated with their zymographic activities.Conclusion. Pulmonary susceptibility to systemic inflammation was highest after LPS, intermediate after CpG-ODN, and lowest after LTA challenge.

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