Tristetraprolin down-regulates IL-23 expression in colon cancer cells

Interleukin 23 (IL-23) is an inflammatory cytokine that plays an important role in tumor promotion. Expression of IL-23 is increased in cancer cells and correlates with tumor progression. However, the mechanisms regulating IL-23 expression in cancer cells are still unclear. Here we report that tristetraprolin (TTP), an AU-rich element (ARE)-binding protein, inhibits IL-23 production in CT26 mouse colon cancer cells. Overexpression of TTP decreased the stability of IL-23 mRNA and the expression level of IL-23 in CT26 cells. Conversely, inhibition of TTP by siRNA increased IL-23 production. TTP destabilized a luciferase mRNA reporter containing the IL-23 mRNA 3’UTR, which contains five AREs. Analyses of deletion and point mutants of the IL-23 mRNA 3’UTR demonstrated that the ARE cluster between the third and fifth AREs was responsible for TTP-mediated destabilization of IL-23 mRNA. A RNA electrophoretic mobility shift assay confirmed that TTP binds to this ARE cluster. Taken together, these results demonstrate that TTP acts as a negative regulator of IL-23 gene expression in mouse colon cancer cells and suggest its potential application as a novel therapeutic target to control IL-23-mediated tumor promotion.     

Time-dependent changes in lipid metabolism in mice with methionine choline deficiency-induced fatty liver disease

Methionine and choline-deficient diet (MCD)-induced fatty liver is one of the best-studied animal models of fatty liver disease. The present study was performed to clarify the relative contributions of individual lipid metabolic pathways to the pathogenesis of MCD-induced fatty liver. Hepatic lipogenesis mediated by the sterol regulatory element-binding protein (SREBP-1c) was increased at 1 week, but not at 6 weeks, of MCD feeding. On the other hand, ¹⁴C-palmitate oxidation did not change at 1 week, but significantly decreased at 6 weeks. This decrease was associated with increased expression of fatty acid translocase, a key enzyme involved in fatty acid uptake. Expression of endoplasmic reticulum stress markers was increased in mice given MCD for both 1 and 6 weeks. These findings suggest the presence of time-dependent differences in lipid metabolism in MCD-induced fatty liver disease: SREBP-1c-mediated lipogenesis is important in the early stages of fatty liver disease, whereas increased fatty acid uptake and decreased fatty acid oxidation become more important in the later stages.     

Isolation of circular yeast artificial chromosomes for synthetic biology and functional genomics studies

Circular yeast artificial chromosomes (YACs) provide significant advantages for cloning and manipulating large segments of genomic DNA in Saccharomyces cerevisiae. However, it has been difficult to exploit these advantages, because circular YACs are difficult to isolate and purify. Here we describe a method for purification of large circular YACs that is more reliable compared with previously described protocols. This method has been used to purify YACs up to 600 kb in size. The purified YAC DNA is suitable for restriction enzyme digestion, DNA sequencing and functional studies. For example, YACs carrying full-size genes can be purified from yeast and used for transfection into mammalian cells or for the construction of a synthetic genome that can be used to produce a synthetic cell. This method for isolating high-quality YAC DNA in microgram quantities should be valuable for functional and synthetic genomic studies. The entire protocol takes ～3 d to complete.     

Natural form of noncytolytic flexible human Fc as a long-acting carrier of agonistic ligand, erythropoietin

Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement-mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc) consisting of IgD and IgG4, and tested its function using erythropoietin (EPO) conjugate, EPO-hyFc. Despite low amino acid homology (20.5%) between IgD Fc and IgG4 Fc, EPO-hyFc retained "Y-shaped" structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FcγR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H), a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H) not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUC(last)). Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach.     

Mobile Phone Use,Blood Lead Levels,and Attention Deficit Hyperactivity Symptoms in Children: A Longitudinal Study

Background

Concerns have developed for the possible negative health effects of radiofrequency electromagnetic field (RF-EMF) exposure to children’s brains. The purpose of this longitudinal study was to investigate the association between mobile phone use and symptoms of Attention Deficit Hyperactivity Disorder (ADHD) considering the modifying effect of lead exposure.

Methods

A total of 2,422 children at 27 elementary schools in 10 Korean cities were examined and followed up 2 years later. Parents or guardians were administered a questionnaire including the Korean version of the ADHD rating scale and questions about mobile phone use, as well as socio-demographic factors. The ADHD symptom risk for mobile phone use was estimated at two time points using logistic regression and combined over 2 years using the generalized estimating equation model with repeatedly measured variables of mobile phone use, blood lead, and ADHD symptoms, adjusted for covariates.

Results

The ADHD symptom risk associated with mobile phone use for voice calls but the association was limited to children exposed to relatively high lead.

Conclusions

The results suggest that simultaneous exposure to lead and RF from mobile phone use was associated with increased ADHD symptom risk, although possible reverse causality could not be ruled out.     

A new integrated method for analyzing heart mechanics using a cell-hemodynamics-autonomic nerve control coupled model of the cardiovascular system

A model of the cardiovascular system coupling cell, hemodynamics, and autonomic nerve control function is proposed for analyzing heart mechanics. We developed a comprehensive cardiovascular model with multi-physics and multi-scale characteristics that simulates the physiological events from membrane excitation of a cardiac cell to contraction of the human heart and systemic blood circulation and ultimately to autonomic nerve control. A lumped parameter model is used to compute the systemic and pulmonary circulations interacting with the cardiac cell mechanism. For autonomic control of the cardiovascular system, we used the approach suggested by Heldt et al. [2002. Computational modeling of cardiovascular response to orthostatic stress. J. Appl. Physiol. 92, 1239–1254] (Heldt model), including baroreflex and cardiopulmonary reflexes. We assumed sympathetic and parasympathetic pathways for the nerve control system. The cardiac muscle response to these reflex control systems was implemented using the activation-level changes in the L-type calcium channel and sarcoplasmic/endoplasmic reticulum calcium ATPase function based on experimental observations. Using this model, we delineated the cellular mechanism of heart contractility mediated by nerve control function. To verify the integrated method, we simulated a 10% hemorrhage, which involves cardiac cell mechanics, circulatory hemodynamics, and nerve control function. The computed and experimental results were compared. Using this methodology, the state of cardiac contractility, influenced by diverse properties such as the afterload and nerve control systems, is easily assessed in an integrated manner.