Measurement of glyoxalase gene expression

2014 ◽  
Vol 42 (2) ◽  
pp. 495-499 ◽  
Author(s):  
Mingzhan Xue ◽  
Naila Rabbani ◽  
Paul J. Thornalley
Keyword(s):  
Gene Expression ◽  
Protein Quantification ◽  
Glyoxalase System ◽  
Protein Levels ◽  
Glyoxalase Ii ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Common Technique ◽  
The Common

The glyoxalase system is an important component of the enzymatic defence against glycation, preventing particularly quantitatively and functionally important glycation of protein and DNA by methylglyoxal. Expression of genes encoding Glo1 (glyoxalase I) and Glo2 (glyoxalase II) may be induced or suppressed, and rates of proteolysis of Glo1 and Glo2 proteins may change in health and disease. Quantitative assessment of glyoxalase gene expression at the mRNA and protein levels has become a key part of glyoxalase system characterization. For mRNA, there is the common technique of real-time RT (reverse transcription)–PCR and direct quantification of mRNA copy number by the Nanostring™ method. For glyoxalase protein quantification, there is the commonly used Western blotting, and also immunoassay and, in proteome-wide studies, quantitative proteomics and proteome dynamics. We provide protocols for the common methods below and briefly review their application.

scholarly journals Regulation of ABCA1 and ABCG1 gene expression in the intraabdominal adipose tissue

2016 ◽  
Vol 62 (3) ◽  
pp. 283-289 ◽  
Author(s):  
V.V. Miroshnikova ◽  
A.A. Panteleeva ◽  
E.A. Bazhenova ◽  
E.P. Demina ◽  
T.S. Usenko ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Cholesterol Metabolism ◽  
Nuclear Factor ◽  
Specific Expression ◽  
Protein Levels ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
And Control

Tissue specific expression of genes encoding cholesterol transporters ABCA1 and ABCG1 as well as genes encoding the most important transcriptional regulators of adipogenesis – LXRa, LXRb, PPARg and RORa has been investigated in intraabdominal adipose tissue (IAT) samples.A direct correlation between the content of ABCA1 and ABCG1 proteins with RORa protein level (r=0.480, p<0.05; r=0.435, p<0.05, respectively) suggests the role of the transcription factor RORa in the regulation of IAT ABCA1 and ABCG1 protein levels. ABCA1 and ABCG1 gene expression positively correlated with obesity indicators such as body mass index (BMI) (r=0.522, p=0.004; r=0.594, p=0.001, respectively) and waist circumference (r=0.403, p=0.033; r=0.474, p=0.013, respectively). The development of obesity is associated with decreased IAT levels of RORa and LXRb mRNA (p=0.016 and p=0.002, respectively). These data suggest that the nuclear factor RORa can play a significant role in the regulation of cholesterol metabolism and control IAT expression of ABCA1 and ABCG1, while the level of IAT LXRb gene expression may be an important factor associated with the development of obesity.

scholarly journals Alterations of mesenchymal stromal cells in cerebrospinal fluid: insights from transcriptomics and an ALS clinical trial

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ashley A. Krull ◽  
Deborah O. Setter ◽  
Tania F. Gendron ◽  
Sybil C. L. Hrstka ◽  
Michael J. Polzin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cerebrospinal Fluid ◽  
Growth Factors ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Large Scale ◽  
Therapeutic Benefit ◽  
Protein Levels ◽  
Genes Encoding ◽  
Intrathecal Space

Abstract Background Mesenchymal stromal cells (MSCs) have been studied with increasing intensity as clinicians and researchers strive to understand the ability of MSCs to modulate disease progression and promote tissue regeneration. As MSCs are used for diverse applications, it is important to appreciate how specific physiological environments may stimulate changes that alter the phenotype of the cells. One need for neuroregenerative applications is to characterize the spectrum of MSC responses to the cerebrospinal fluid (CSF) environment after their injection into the intrathecal space. Mechanistic understanding of cellular biology in response to the CSF environment may predict the ability of MSCs to promote injury repair or provide neuroprotection in neurodegenerative diseases. Methods In this study, we characterized changes in morphology, metabolism, and gene expression occurring in human adipose-derived MSCs cultured in human (hCSF) or artificial CSF (aCSF) as well as examined relevant protein levels in the CSF of subjects treated with MSCs for amyotrophic lateral sclerosis (ALS). Results Our results demonstrated that, under intrathecal-like conditions, MSCs retained their morphology, though they became quiescent. Large-scale transcriptomic analysis of MSCs revealed a distinct gene expression profile for cells cultured in aCSF. The aCSF culture environment induced expression of genes related to angiogenesis and immunomodulation. In addition, MSCs in aCSF expressed genes encoding nutritional growth factors to expression levels at or above those of control cells. Furthermore, we observed a dose-dependent increase in growth factors and immunomodulatory cytokines in CSF from subjects with ALS treated intrathecally with autologous MSCs. Conclusions Overall, our results suggest that MSCs injected into the intrathecal space in ongoing clinical trials remain viable and may provide a therapeutic benefit to patients.

scholarly journals Selection of reference genes for gene expression analysis in Liriodendron hybrids’ somatic embryogenesis and germinative tissues

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tingting Li ◽  
Weigao Yuan ◽  
Shuai Qiu ◽  
Jisen Shi
Keyword(s):  
Gene Expression ◽  
Somatic Embryogenesis ◽  
Reference Genes ◽  
Gene Selection ◽  
Elongation Factor ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Suitable Reference ◽  
Functional Analyses ◽  
Elongation Factor 1

AbstractThe differential expression of genes is crucial for plant somatic embryogenesis (SE), and the accurate quantification of gene expression levels relies on choosing appropriate reference genes. To select the most suitable reference genes for SE studies, 10 commonly used reference genes were examined in synchronized somatic embryogenic and subsequent germinative cultures of Liriodendron hybrids by using quantitative real-time reverse transcription PCR. Four popular normalization algorithms: geNorm, NormFinder, Bestkeeper and Delta-Ct were used to select and validate the suitable reference genes. The results showed that elongation factor 1-gamma, histone H1 linker protein, glyceraldehyde-3-phosphate dehydrogenase and α-tubulin were suitable for SE tissues, while elongation factor 1-gamma and actin were best for the germinative organ tissues. Our work will benefit future studies of gene expression and functional analyses of SE in Liriodendron hybrids. It is also serves as a guide of reference gene selection in early embryonic gene expression analyses for other woody plant species.

scholarly journals Alopecia in Harlequin mutant mice is associated with reduced AIF protein levels and expression of retroviral elements

2020 ◽  
Author(s):  
Maik Hintze ◽  
Sebastian Griesing ◽  
Marion Michels ◽  
Birgit Blanck ◽  
Lena Wischhof ◽  
...  
Keyword(s):  
Hair Growth ◽  
Transcriptional Regulators ◽  
Mutant Mice ◽  
Wild Type ◽  
Protein Levels ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Keratinocyte Cell ◽  
Apoptosis Inducing Factor ◽  
Hair Cortex

AbstractWe investigated the contribution of apoptosis-inducing factor (AIF), a key regulator of mitochondrial biogenesis, in supporting hair growth. We report that pelage abnormalities developed during hair follicle (HF) morphogenesis in Harlequin (Hq) mutant mice. Fragility of the hair cortex was associated with decreased expression of genes encoding structural hair proteins, though key transcriptional regulators of HF development were expressed at normal levels. Notably, Aifm1 (R200 del) knockin males and Aifm1(R200 del)/Hq females showed minor hair defects, despite substantially reduced AIF levels. Furthermore, we cloned the integrated ecotropic provirus of the Aifm1Hq allele. We found that its overexpression in wild-type keratinocyte cell lines led to down-regulation of HF-specific Krt84 and Krtap3-3 genes without altering Aifm1 or epidermal Krt5 expression. Together, our findings imply that pelage paucity in Hq mutant mice is mechanistically linked to severe AIF deficiency and is associated with the expression of retroviral elements that might potentially influence the transcriptional regulation of structural hair proteins.

scholarly journals Variable Expressions of Staphylococcus aureus Bicomponent Leucotoxins Semiquantified by Competitive Reverse Transcription-PCR

2000 ◽  
Vol 66 (9) ◽  
pp. 3931-3938 ◽  
Author(s):  
St�phane Bronner ◽  
Patricia Stoessel ◽  
Alain Gravet ◽  
Henri Monteil ◽  
Gilles Pr�vost
Keyword(s):  
Staphylococcus Aureus ◽  
Reverse Transcription ◽  
Regulatory System ◽  
Optimization Procedure ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Specific Mrnas ◽  
Casamino Acids ◽  
Global Regulatory System

ABSTRACT A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of Staphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemolysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/104 CFU to 102 mRNA/CFU, depending on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of hlgA expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxin-associated virulence. The agr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system inS. aureus, except that expression of hlgA was not affected in the agr mutant.

scholarly journals Efflux-Related Resistance to Norfloxacin, Dyes, and Biocides in Bloodstream Isolates of Staphylococcus aureus

2007 ◽  
Vol 51 (9) ◽  
pp. 3235-3239 ◽  
Author(s):  
Carmen E. DeMarco ◽  
Laurel A. Cushing ◽  
Emmanuel Frempong-Manso ◽  
Susan M. Seo ◽  
Tinevimbo A. A. Jaravaza ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Efflux Pump Inhibitor ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Environmental Surfaces ◽  
Genes Encoding ◽  
High Level

ABSTRACT Efflux is an important resistance mechanism in Staphylococcus aureus, but its frequency in patients with bacteremia is unknown. Nonreplicate bloodstream isolates were collected over an 8-month period, and MICs of four common efflux pump substrates, with and without the broad-spectrum efflux pump inhibitor reserpine, were determined (n = 232). A reserpine-associated fourfold decrease in MIC was considered indicative of efflux. Strains exhibiting efflux of at least two of the four substrates were identified (“effluxing strains” [n = 114]). For these strains, MICs with or without reserpine for an array of typical substrates and the expression of mepA, mdeA, norA, norB, norC, and qacA/B were determined using quantitative real-time reverse transcription-PCR (qRT-PCR). A fourfold or greater increase in gene expression was considered significant. The most commonly effluxed substrates were ethidium bromide and chlorhexidine (100 and 96% of effluxing strains, respectively). qRT-PCR identified strains overexpressing mepA (5 [4.4%]), mdeA (13 [11.4%]), norA (26 [22.8%]), norB (29 [25.4%]), and norC (19 [16.7%]); 23 strains overexpressed two or more genes. Mutations probably associated with increased gene expression included a MepR-inactivating substitution and norA promoter region insertions or deletions. Mutations possibly associated with increased expression of the other analyzed genes were also observed. Effluxing strains comprised 49% of all strains studied (114/232 strains), with nearly half of these overexpressing genes encoding MepA, MdeA, and/or NorABC (54/114 strains). Reduced susceptibility to biocides may contribute to persistence on environmental surfaces, and efflux of drugs such as fluoroquinolones may predispose strains to high-level target-based resistance.

scholarly journals Expression of the apoptosis-releated genes in patients with newly diagnosed chronic lymphocytic leukemia in clinical data context

2018 ◽  
Vol 17 (2) ◽  
pp. 41-46 ◽  
Author(s):  
S. G. Zakharov ◽  
A. K. Golenkov ◽  
A. V. Misyurin ◽  
E. V. Kataeva ◽  
A. A. Rudakova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Manifestations ◽  
Multivariate Regression Analysis ◽  
Lymphocytic Leukemia ◽  
Newly Diagnosed ◽  
Expression Of Genes ◽  
Gene Level ◽  
Genes Encoding ◽  
Group Ii ◽  
High Level

Introduction.The given data of fundamental studies of apoptosis processes in B-cell lymphocytic leukemia (B-CLL) testifies about the complexity and variety of mechanisms affecting the kinetics of normal cells and tumor lymphocytes in this disease. It is important to study the severity of clinical manifestations of the disease depending on the expression of the genes that modulate apoptosis.The purposeof the study is to compare the activity of genes encoding apoptosis modulators, the cell cycle and cancer-testicular PRAME protein with clinical manifestations of the disease in primary patients with B-CLL.Materials and methods.The level of expression of the proapoptotic genes FAS, TRAIL, TNFR2, DR4/5 and DR3, as well as the HSP27, XIAP genes, blocking apoptosis was determined in 23 patients with newly diagnosed chronic B-CLL. In addition, expression of genes TP53 and P21 and cancer-testis gene PRAME are tested.Results.According to the multivariate regression analysis, the FAS gene expression in the onset of the disease had the greatest impact on the clinical characteristics of the disease. In this connection, the patients were divided into groups with normal (group) and low gene level (group II). A low level of FAS expression (Me 387 %) was associated with stage II disease (p = 0.03), a large number of lympho cytes (p = 0.001), fewer erythrocytes (p = 0.08), and a lower level of TNFR2 gene expression (p = 0.08), high level of expression of XIAP, HSP27, P21. Overall, the anti-apoptotic potential in Group II patients was higher, which was accompanied by more pronounced clinical manifestations of the disease.Conclusions.The increased anti-apoptotic potential of tumor lymphocytes in newly diagnosed B-CLL is accompanied by a larger tumor mass and greater clinical and hematological manifestation of the disease.

Abstract 191: Transcriptional Regulation of Renin by Nuclear Receptors Co-regulated With Renin

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Ko-Ting Lu ◽  
Eric T Weatherford ◽  
Pimonrat Ketsawatsomkron ◽  
Justin L Grobe ◽  
Curt D Sigmund
Keyword(s):  
Gene Expression ◽  
Nuclear Receptors ◽  
Estrogen Receptor Alpha ◽  
Hormone Receptors ◽  
Cell Types ◽  
Negative Control ◽  
Sirna Duplex ◽  
Expression Of Genes ◽  
Renin Gene ◽  
Genes Encoding

Expression of the renin gene is required to maintain normal morphological and physiological identity of renal juxtaglomerular (JG) cells, yet the mechanisms regulating renin gene transcription remain elusive. We re-examined data from Brunskill et. al (JASN 22:2213, 2011), investigating genome-wide gene expression in JG and other renal cell types. Based on our previous data implicating nuclear receptors (RAR, RXR, VDR, PPARG, Nr2f2 and Nr2f6) in the regulation of mouse and human renin gene expression, we focused our analysis on the expression of genes encoding the 48 nuclear hormone receptors and their co-regulation with renin. Several nuclear receptors have an expression pattern emulating that of renin, that is, they were similarly enriched in JG cells but not in other cell types. These include Esr1, Nr1h4, Ppara, VDR, Nr1i2, Ppard, Hnf4g, Nr1h3, Thrb, Hnf4a, Esrrg, Nr4a3, Nr3c2, and Ar. We tested the hypothesis that a nuclear receptor that is co-regulated with renin may participate in renin gene regulation. To accomplish this, endogenous renin expression was evaluated in renin-expressing As4.1 cells after siRNA-mediated knock down of selected nuclear receptors. Each experiment included a negative control siRNA duplex (NC) that does not target any known genes. By way of example, siRNA-mediated inhibition of estrogen receptor alpha (Esr1) by 70-80% resulted in a 2-fold decrease in renin mRNA (fold change ± SEM: siEsr1: 0.4±0.2, p<0.001 vs NC). Similar results were obtained with a different siRNA targeting Esr1. Interestingly, loss of Esr1 also caused up-regulation of vitamin D receptor (VDR, 2.8±0.7 fold, p<0.001 vs NC) and Nr2f6 (2.0±0.2 fold, p<0.05 vs NC), both of which are known to be negative regulators of renin. Similarly, both renin (0.1±0.02, p<0.001 vs untreated) and Esr1 (0.3±0.1, p<0.05 vs untreated) mRNA were reduced in the kidney from mice treated with deoxycorticosterone acetate (50mg) and receiving 0.15 M NaCl in drinking water for 21 days (DOCA-salt). These data suggest Esr1 may regulate renin expression. Studies are in progress to assess if Esr1 stimulates renin expression on its own or acts by affecting the level of other nuclear receptors; and to determine if other co-regulated nuclear receptors also regulate expression of the renin gene.

scholarly journals Up-Regulation Mttp and Apob Gene Expression in Rat Liver is Related to Post-Lipectomy Hypertriglyceridemia

2015 ◽  
Vol 36 (5) ◽  
pp. 1767-1777 ◽  
Author(s):  
Agnieszka Dettlaff-Pokora ◽  
Tomasz Sledzinski ◽  
Julian Swierczynski
Keyword(s):  
Gene Expression ◽  
Fatty Acid Synthase ◽  
Protein A ◽  
Surgical Removal ◽  
Gene Expressions ◽  
Transfer Protein ◽  
Expression Of Genes ◽  
Spot 14 ◽  
Genes Encoding ◽  
Experimental Rat Model

Background/Aims: The aim of this study was to explain the molecular basis for elevated concentrations of circulating triglycerides (TAGs) after partial surgical removal of adipose tissue (lipectomy) in rats. Methods: The levels of mRNA and protein: a) involved in synthesis of fatty acids and TAGs; b) participating in TAG-rich lipoproteins assembly and secretion; and c) transcription factors essential for maintaining TAG homeostasis were determined by RT-PCR and Western Blot in the livers of control and lipectomized rats. Results: Partial lipectomy was associated with increase: a) in serum and liver concentration of TAGs, and b) in the liver levels of mRNA of microsomal TAG transfer protein (MTP) and apolipoprotein B-100 (ApoB-100). These changes were tightly associated with up-regulation of Hnf1a and Hnf4a gene expression in the liver. Lipectomy was also reflected by a significant increase in the expression of genes encoding: a) fatty acid synthase (FASN), b) glycerol 3-phosphate acyltransferase 1 (GPAT1), diacylglycerol acyltransferases 1 and 2 (DGAT1 and DGAT2), c) spot 14 protein (S14) and SREBP-1 in the liver. Conclusion: Coordinated up-regulation of Mttp, Apob, Hnf1a, Hnf4a, Fasn, Gpam and Dgat (1 and 2) gene expressions may contribute to the increase in circulating and liver concentrations of TAGs after lipectomy in an experimental rat model.

scholarly journals Irisin Recovers Osteoarthritic Chondrocytes In Vitro

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1478 ◽  
Author(s):  
Gianluca Vadalà ◽  
Giuseppina Di Giacomo ◽  
Luca Ambrosio ◽  
Francesca Cannata ◽  
Claudia Cicione ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Protein Quantification ◽  
Saline Control ◽  
Collagen Gene ◽  
Type Ii ◽  
Protein Levels ◽  
Collagen Gene Expression ◽  
Osteoarthritic Chondrocytes

Physical exercise favors weight loss and ameliorates articular pain and function in patients suffering from osteoarthritis. Irisin, a myokine released upon muscle contraction, has demonstrated to yield anabolic effects on different cell types. This study aimed to investigate the effect of irisin on human osteoarthritic chondrocytes (hOAC) in vitro. Our hypothesis was that irisin would improve hOAC metabolism and proliferation. Cells were cultured in growing media and then exposed to either phosphate-buffered saline (control group) or human recombinant irisin (experimental group). Cell proliferation, glycosaminoglycan content, type II/X collagen gene expression and protein quantification as well as p38/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK), protein kinase B (Akt), c-Jun N-terminal kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) involvement were evaluated. Furthermore, gene expression of interleukin (IL)-1 and -6, matrix metalloproteinase (MMP)-1 and -13, inducible nitric oxide synthase (iNOS), and tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -3 were investigated following irisin exposure. Irisin increased hOAC cell content and both type II collagen gene expression and protein levels, while decreased type X collagen gene expression and protein levels. Moreover, irisin decreased IL-1, IL-6, MMP-1, MMP-13 and iNOS gene expression, while increased TIMP-1 and TIMP-3 levels. These effects seemed to be mediated by inhibition of p38, Akt, JNK and NFκB signaling pathways. The present study suggested that irisin may stimulate hOAC proliferation and anabolism inhibiting catabolism through p38, Akt, JNK, and NFκB inactivation in vitro, demonstrating the existence of a cross-talk between muscle and cartilage.

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