Evaluation of sterol­o­acyl transferase 1 and cholesterol ester levels in plasma, peritoneal fluid and tumor tissue of patients with endometrial cancer: A pilot study.

Endometrial cancer (EC) is the most prevalent gynecological malignancy. Abnormal accumulation of sterol-O-acyl transferase 1 (SOAT1) and SOAT1-mediated cholesterol ester (CE) contributes to cancer progression in various malignancies, including ovarian cancer. Therefore, it was hypothesized that similar molecular changes may occur in EC. The present study aimed to evaluate the diagnostic and/or prognostic potential of SOAT1 and CE in EC by: i) Determining SOAT1 and CE levels in plasma, peritoneal fluid and endometrial tissue from patients with EC and control subjects; ii) performing receiver operating characteristic curve analysis to determine diagnostic performance; iii) comparing SOAT1 and CE expression to that of the tumor proliferation marker Ki67; and iv) assessing the association between SOAT1 expression and survival. Enzyme-linked immunosorbent assay was used to determine the levels of SOAT1 protein in tissue, plasma and peritoneal fluid. The mRNA and protein expression levels of SOAT1 and Ki67 in tissues were detected by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. CE levels were determined colorimetrically in plasma and peritoneal fluid. SOAT1-associated survival data from the cBioPortal cancer genomics database were used to assess prognostic relevance. The results revealed that SOAT1 and CE levels were significantly elevated in tumor tissue and peritoneal fluid samples collected from the EC group. By contrast, the plasma levels of SOAT1 and CE in the EC and control groups were similar. Significant positive associations between CE and SOAT1, SOAT1/CE and Ki67, and SOAT1/CE and poor overall survival in patients with EC suggested that SOAT1/CE may be associated with malignancy, aggressiveness and poor prognosis. In conclusion, SOAT1 and CE may serve as potential biomarkers for prognosis and target-specific treatment of EC.

Assessment of the diagnostic and prognostic relevance of ACAT1 and CE levels in plasma, peritoneal fluid and tumor tissue of epithelial ovarian cancer patients - a pilot study.

BACKGROUND: Abnormal accumulation of acyl-CoA cholesterol acyltransferase-1 (ACAT1) and ACAT1-mediated cholesterol esterified with fatty acids (CE) contribute to cancer progression in various cancers. Our findings of increased CE and ACAT1 levels in epithelial ovarian cancer (EOC) cell lines prompted us to investigate whether such an increase occurs in primary clinical samples obtained from human subjects diagnosed with EOC. We evaluated the diagnostic/prognostic potential of ACAT1 and CE in EOC by: 1) assessing ACAT1 and CE levels in plasma, peritoneal fluid, and ovarian/tumor tissues; 2) assessing diagnostic performance by Receiver Operating Characteristic (ROC) analysis; and 3) comparing expression of ACAT1 and CE with that of tumor proliferation marker, Ki67. METHODS: ACAT1 protein levels in plasma, peritoneal fluid and tissue were measured via enzyme-linked immunosorbent assay. Tissue expression of ACAT1 and Ki67 proteins were confirmed by immunohistochemistry and mRNA transcript levels were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). CE levels were assessed in plasma, peritoneal fluid (colorimetric assay) and in tissue (thin layer chromatography). RESULTS: Preoperative levels of ACAT1 and CE on the day of surgery were significantly higher in tissue and peritoneal fluid from EOC patients vs. the non-malignant group, which included subjects with benign tumors and normal ovaries; however, no significant differences were observed in plasma. In tissue and peritoneal fluid, positive correlations were observed between CE and ACAT1 levels, as well as between ACAT1/CE and Ki67. CONCLUSIONS: ACAT1 and CE accumulation may be linked to the aggressive potential of EOC; therefore, these mediators may be useful biomarkers for EOC prognosis and target-specific treatments.

Early history of circular RNAs, children of splicing.

In this commentary we briefly summarize early work on circular RNAs derived from spliceosome mediated circularization. We highlight how this early work inspired work on the basic mechanisms of nuclear RNA splicing, the possible function of circular RNAs and the potential uses of circular RNAs as tools in biomedicine. Recent developments in the study of circular RNAs, summarized in this volume, have brought these questions back to the foreground.

Substrate specificity and catalysis by the editing active site of Alanyl-tRNA synthetase from Escherichia coli.

Aminoacyl-tRNA synthetases (ARSs) enhance the fidelity of protein synthesis through multiple mechanisms, including hydrolysis of the adenylate and cleavage of misacylated tRNA. Alanyl-tRNA synthetase (AlaRS) limits misacylation with glycine and serine by use of a dedicated editing domain, and a mutation in this activity has been genetically linked to a mouse model of a progressive neurodegenerative disease. Using the free-standing Pyrococcus horikoshii AlaX editing domain complexed with serine as a model and both Ser-tRNA(Ala) and Ala-tRNA(Ala) as substrates, the deacylation activities of the wild type and five different Escherichia coli AlaRS editing site substitution mutants were characterized. The wild-type AlaRS editing domain deacylated Ser-tRNA(Ala) with a k(cat)/K(M) of 6.6 × 10(5) M(-1) s(-1), equivalent to a rate enhancement of 6000 over the rate of enzyme-independent deacylation but only 12.2-fold greater than the rate with Ala-tRNA(Ala). While the E664A and T567G substitutions only minimally decreased k(cat)/K(M,) Q584H, I667E, and C666A AlaRS were more compromised in activity, with decreases in k(cat)/K(M) in the range of 6-, 6.6-, and 15-fold. C666A AlaRS was 1.7-fold more active on Ala-tRNA(Ala) relative to Ser-tRNA(Ala), providing the only example of a true reversal of substrate specificity and highlighting a potential role of the coordinated zinc in editing substrate specificity. Along with the potentially serious physiological consequences of serine misincorporation, the relatively modest specificity of the AlaRS editing domain may provide a rationale for the widespread phylogenetic distribution of AlaX free-standing editing domains, thereby contributing a further mechanism to lower concentrations of misacylated tRNA(Ala).

Monitoring RNA transcription in real time by using surface plasmon resonance.

The decision to elongate or terminate the RNA chain at specific DNA template positions during transcription is kinetically regulated, but the methods used to measure the rates of these processes have not been sufficiently quantitative to permit detailed mechanistic analysis of the steps involved. Here, we use surface plasmon resonance (SPR) technology to monitor RNA transcription by Escherichia coli RNA polymerase (RNAP) in solution and in real time. We show that binding of RNAP to immobilized DNA templates to form active initiation or elongation complexes can be resolved and monitored by this method, and that changes during transcription that involve the gain or loss of bound mass, including the release of the sigma factor during the initiation-elongation transition, the synthesis of the RNA transcript, and the release of core RNAP and nascent RNA at intrinsic terminators, can all be observed. The SPR method also permits the discrimination of released termination products from paused and other intermediate complexes at terminators. We have used this approach to show that the rate constant for transcript release at intrinsic terminators tR2 and tR' is approximately 2-3 s(-1) and that the extent of release at these terminators is consistent with known termination efficiencies. Simulation techniques have been used to fit the measured parameters to a simple kinetic model of transcription and the implications of these results for transcriptional regulation are discussed.

Reaction pathways in transcript elongation.

Transcription of DNA into RNA is a central part of gene expression, and is highly regulated in all organisms. In order to approach transcription control systems on a molecular basis we must understand the mechanisms used by the transcription complex to discharge its various functions, which include transcript initiation, elongation, editing, and termination. In this article we describe recent progress in sorting out the multiple reaction pathways that are, at least in principle, available to the transcription complex at each DNA template position, and show how transcription control systems partition active complexes into these pathways. Understanding these regulatory processes requires an elucidation of the molecular details of how sequence- and factor-dependent changes in the conformations, stabilities, and reaction rates of the complexes determine function. Recent progress in unraveling these issues is summarized in this article and emerging principles that govern the regulation of the elongation phase of transcription are discussed.

Active Escherichia coli transcription elongation complexes are functionally homogeneous.

The elongation phase of RNA transcription represents a major target for the regulation of gene expression. Two general classes of models have been proposed to define the dynamic properties of transcription complexes in the elongation phase. Stable heterogeneity models posit that the ensemble of active elongation-competent complexes consists of multiple distinct and stable forms that are specified early in the transcription cycle and isomerize to other forms slowly. In contrast, equilibrium or rapid interconversion models require that active elongation complexes interconvert rapidly on the time-scale of single nucleotide addition. Measurements of transcription termination efficiency (TE) can be used to distinguish between these models, because stable heterogeneity models predict that the termination-resistant fraction of an elongation complex population should be enriched after transcription through an upstream terminator, leading to a decreased TE at downstream terminators. In contrast, rapid interconversion models require that the population of active (elongation-competent) complexes equilibrate after transcription through each terminator and, therefore, that the value of TE observed at identical upstream and downstream terminators should be the same. We have constructed transcription templates containing multiple identical terminators and found no significant changes in TE with terminator position along the template. Various other forms of upstream treatment of elongation complex populations also were used to attempt to fractionate the complexes into functionally different forms. None of these treatments changed the apparent TE at downstream terminators. These results are consistent with a rapid interconversion model of transcript elongation. The consequences of these results for the regulation of gene expression are discussed.