Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

<p>Abstract</p> <p>Background</p> <p>Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Quantitative real-time PCR (RT qPCR) is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB.</p> <p>Results</p> <p>The present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], β-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA]) in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan^®Gene Expression Assays). Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis.</p> <p>Conclusion</p> <p>This study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects.</p

Sequential poly-ubiquitylation by specialized conjugating enzymes expands the versatility of a quality control ubiquitin ligase

The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48-linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the number of ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility yet sustains the specificity of the Ub conjugation system

ZnT3 mRNA levels are reduced in Alzheimer's disease post-mortem brain

<p>Abstract</p> <p>Background</p> <p>ZnT3 is a membrane Zn²⁺transporter that is responsible for concentrating Zn²⁺into neuronal presynaptic vesicles. Zn²⁺homeostasis in the brain is relevant to Alzheimer's disease (AD) because Zn²⁺released during neurotransmission may bind to Aβ peptides, accelerating the assembly of Aβ into oligomers which have been shown to impair synaptic function.</p> <p>Results</p> <p>We quantified ZnT3 mRNA levels in Braak-staged human post mortem (pm) brain tissue from medial temporal gyrus, superior occipital gyrus, superior parietal gyrus, superior frontal gyrus and cerebellum from individuals with AD (n = 28), and matched controls (n = 5) using quantitative real-time PCR. ZnT3 mRNA levels were significantly decreased in all four cortical regions examined in the AD patients, to 45-60% of control levels. This reduction was already apparent at Braak stage 4 in most cortical regions examined. Quantification of neuronal and glial-specific markers in the same samples (neuron-specific enolase, NSE; and glial fibrillary acidic protein, GFAP) indicated that loss of cortical ZnT3 expression was more pronounced, and occurred prior to, significant loss of NSE expression in the tissue. Significant increases in cortical GFAP expression were apparent as the disease progressed. No gene expression changes were observed in the cerebellum, which is relatively spared of AD neuropathology.</p> <p>Conclusions</p> <p>This first study to quantify ZnT3 mRNA levels in human pm brain tissue from individuals with AD and controls has revealed a significant loss of ZnT3 expression in cortical regions, suggesting that neuronal cells in particular show reduced expression of ZnT3 mRNA in the disease. This suggests that altered neuronal Zn²⁺handling may be an early event in AD pathogenesis.</p

Tissue-derived proinflammatory effect of adenosine A2B receptor in lung ischemia–reperfusion injury

ObjectiveIschemia–reperfusion injury after lung transplantation remains a major source of morbidity and mortality. Adenosine receptors have been implicated in both pro- and anti-inflammatory roles in ischemia–reperfusion injury. This study tests the hypothesis that the adenosine A2B receptor exacerbates the proinflammatory response to lung ischemia–reperfusion injury.MethodsAn in vivo left lung hilar clamp model of ischemia–reperfusion was used in wild-type C57BL6 and adenosine A2B receptor knockout mice, and in chimeras created by bone marrow transplantation between wild-type and adenosine A2B receptor knockout mice. Mice underwent sham surgery or lung ischemia–reperfusion (1 hour ischemia and 2 hours reperfusion). At the end of reperfusion, lung function was assessed using an isolated buffer-perfused lung system. Lung inflammation was assessed by measuring proinflammatory cytokine levels in bronchoalveolar lavage fluid, and neutrophil infiltration was assessed via myeloperoxidase levels in lung tissue.ResultsCompared with wild-type mice, lungs of adenosine A2B receptor knockout mice were significantly protected after ischemia–reperfusion, as evidenced by significantly reduced pulmonary artery pressure, increased lung compliance, decreased myeloperoxidase, and reduced proinflammatory cytokine levels (tumor necrosis factor-α; interleukin-6; keratinocyte chemoattractant; regulated on activation, normal T-cell expressed and secreted; and monocyte chemotactic protein-1). Adenosine A2B receptor knockout→adenosine A2B receptor knockout (donor→recipient) and wild-type→ adenosine A2B receptor knockout, but not adenosine A2B receptor knockout→wild-type, chimeras showed significantly improved lung function after ischemia–reperfusion.ConclusionsThese results suggest that the adenosine A2B receptor plays an important role in mediating lung inflammation after ischemia–reperfusion by stimulating cytokine production and neutrophil chemotaxis. The proinflammatory effects of adenosine A2B receptor seem to be derived by adenosine A2B receptor activation primarily on resident pulmonary cells and not bone marrow-derived cells. Adenosine A2B receptor may provide a therapeutic target for prevention of ischemia–reperfusion-related graft dysfunction in lung transplant recipients

GATA transcription factors drive initial Xist upregulation after fertilization through direct activation of a distal enhancer element

To ensure dosage compensation for X-linked genes between the sexes, one X chromosome is silenced during early embryonic development of female mammals. This process of X-chromosome inactivation (XCI) is initiated through upregulation of the RNA Xist from one X chromosome shortly after fertilization. Xist then mediates chromosome-wide gene silencing in cis and remains expressed in all cell types except the germ line and the pluripotent state, where XCI is reversed. The factors that drive Xist upregulation and thereby initiate XCI remain however unknown. We identify GATA transcription factors as potent Xist activators and demonstrate that they are essential for the activation of Xist in mice following fertilization. Through a pooled CRISPR activation screen we find that GATA1 can drive ectopic Xist expression in murine embryonic stem cells (mESCs). We demonstrate that all GATA factors can activate Xist directly via a GATA-responsive regulatory element (RE79) positioned 100 kb upstream of the Xist promoter. Additionally, GATA factors are essential for the induction of XCI in mouse preimplantation embryos, as simultaneous deletion of three members of the GATA family (GATA1/4/6) in mouse zygotes effectively prevents Xist upregulation. Thus, initiation of XCI and possibly its maintenance in distinct lineages of the preimplantation embryo is ensured by the combined activity of different GATA family members, and the absence of GATA factors in the pluripotent state likely contributes to X reactivation. We thus describe a form of regulation in which the combined action of numerous tissue-specific factors can achieve near-ubiquitous expression of a target gene

Reno-protective effects of renin–angiotensin system blockade in type 2 diabetic patients: a systematic review and network meta-analysis

AIMS/HYPOTHESIS: This meta-analysis aimed to compare the renal outcomes between ACE inhibitor (ACEI)/angiotensin II receptor blocker (ARB) and other antihypertensive drugs or placebo in type 2 diabetes. METHODS: Publications were identified from Medline and Embase up to July 2011. Only randomised controlled trials comparing ACEI/ARB monotherapy with other active drugs or placebo were eligible. The outcome of end-stage renal disease, doubling of serum creatinine, microvascular complications, microalbuminuria, macroalbuminuria and albuminuria regression were extracted. Risk ratios were pooled using a random-effects model if heterogeneity was present; a fixed-effects model was used in the absence of heterogeneity. RESULTS: Of 673 studies identified, 28 were eligible (n = 13-4,912). In direct meta-analysis, ACEI/ARB had significantly lower risk of serum creatinine doubling (pooled RR = 0.66 [95% CI 0.52, 0.83]), macroalbuminuria (pooled RR = 0.70 [95% CI 0.50, 1.00]) and albuminuria regression (pooled RR 1.16 [95% CI 1.00, 1.39]) than other antihypertensive drugs, mainly calcium channel blockers (CCBs). Although the risks of end-stage renal disease and microalbuminuria were lower in the ACEI/ARB group (pooled RR 0.82 [95% CI 0.64, 1.05] and 0.84 [95% CI 0.61, 1.15], respectively), the differences were not statistically significant. The ACEI/ARB benefit over placebo was significant for all outcomes except microalbuminuria. A network meta-analysis detected significant treatment effects across all outcomes for both active drugs and placebo comparisons. CONCLUSIONS/INTERPRETATION: Our review suggests a consistent reno-protective effect of ACEI/ARB over other antihypertensive drugs, mainly CCBs, and placebo in type 2 diabetes. The lack of any differences in BP decrease between ACEI/ARB and active comparators suggest this benefit is not due simply to the antihypertensive effect

Expression profiles of acute lymphoblastic and myeloblastic leukemias with ALL-1 rearrangements

The ALL-1 gene is directly involved in 5-10% of ALLs and AMLs by fusion to other genes or through internal rearrangements. DNA microarrays were utilized to determine expression profiles of ALLs and AMLs with ALL-1 rearrangements. These profiles distinguish those tumors from other ALLs and AMLs. The expression patterns of ALL-1-associated tumors, in particular ALLs, involve oncogenes, tumor suppressors, anti apoptotic genes, drug resistance genes etc., and correlate with the aggressive nature of the tumors. The genes whose expression differentiates between ALLs with and without ALL-1 rearrangement were further divided into several groups enabling separation of ALL-1- associated ALLs into two subclasses. Further, AMLs with partial duplication of ALL-1 vary in their expression pattern from AMLs in which ALL-1 had undergone fusion to other genes. The extensive analysis described here draws attention to genes which might have a direct role in pathogenesis

Functional Diversity and Structural Disorder in the Human Ubiquitination Pathway

The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred - E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well as the mechanism of ubiquitin transfer by long-range conformational transitions. © 2013 Bhowmick et al

Aberrant substrate engagement of the ER translocon triggers degradation by the Hrd1 ubiquitin ligase

Little is known about quality control of proteins that aberrantly or persistently engage the endoplasmic reticulum (ER)-localized translocon en route to membrane localization or the secretory pathway. Hrd1 and Doa10, the primary ubiquitin ligases that function in ER-associated degradation (ERAD) in yeast, target distinct subsets of misfolded or otherwise abnormal proteins based primarily on degradation signal (degron) location. We report the surprising observation that fusing Deg1, a cytoplasmic degron normally recognized by Doa10, to the Sec62 membrane protein rendered the protein a Hrd1 substrate. Hrd1-dependent degradation occurred when Deg1-Sec62 aberrantly engaged the Sec61 translocon channel and underwent topological rearrangement. Mutations that prevent translocon engagement caused a reversion to Doa10-dependent degradation. Similarly, a variant of apolipoprotein B, a protein known to be cotranslocationally targeted for proteasomal degradation, was also a Hrd1 substrate. Hrd1 therefore likely plays a general role in targeting proteins that persistently associate with and potentially obstruct the translocon

Cigarette Smoke Affects Keratinocytes SRB1 Expression and Localization via H2O2 Production and HNE Protein Adducts Formation

Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol uptake. Stratum corneum (SC), the outermost layer of the skin, is composed of more than 25% cholesterol. Several reports support the view that alteration of SC lipid composition may be the cause of impaired barrier function which gives rise to several skin diseases. For this reason the regulation of the genes involved in cholesterol uptake is of extreme significance for skin health. Being the first shield against external insults, the skin is exposed to several noxious substances and among these is cigarette smoke (CS), which has been recently associated with various skin pathologies. In this study we first have shown the presence of SR-B1 in murine and human skin tissue and then by using immunoblotting, immunoprecipitation, RT-PCR, and confocal microscopy we have demonstrated the translocation and the subsequent lost of SR-B1 in human keratinocytes (cell culture model) after CS exposure is driven by hydrogen peroxide (H2O2) that derives not only from the CS gas phase but mainly from the activation of cellular NADPH oxidase (NOX). This effect was reversed when the cells were pretreated with NOX inhibitors or catalase. Furthermore, CS caused the formation of SR-B1-aldheydes adducts (acrolein and 4-hydroxy-2-nonenal) and the increase of its ubiquitination, which could be one of the causes of SR-B1 loss. In conclusion, exposure to CS, through the production of H2O2, induced post-translational modifications of SR-B1 with the consequence lost of the receptor and this may contribute to the skin physiology alteration as a consequence of the variation of cholesterol uptake