The autoregulatory translational control element of poly(A)-binding protein mRNA forms a heteromeric ribonucleoprotein complex

Repression of poly(A)-binding protein (PABP) mRNA translation involves the binding of PABP to the adenine-rich autoregulatory sequence (ARS) in the 5′-untranslated region of its own mRNA. In this report, we show that the ARS forms a complex in vitro with PABP, and two additional polypeptides of 63 and 105 kDa. The 63 and 105 kDa polypeptides were identified, as IMP1, an ortholog of chicken zip-code binding polypeptide, and UNR, a PABP binding polypeptide, respectively, by mass spectrometry of the ARS RNA affinity purified samples. Using a modified ribonucleoprotein (RNP) immunoprecipitation procedure we further show that indeed, both IMP1 and UNR bind to the ARS containing reporter RNA in vivo. Although both IMP1 and UNR could bind independently to the ARS RNA in vitro, their RNA-binding ability was stimulated by PABP. Mutational analyses of the ARS show that the presence of four of the six oligo(A) regions of the ARS was sufficient to repress translation and the length of the conserved pyrimidine spacers between the oligo(A) sequences was important for ARS function. The ability of mutant ARS RNAs to form the PABP, IMP1 and UNR containing RNP complex correlates well with the translational repressor activity of the ARS. There is also a direct relationship between the length of the poly(A) RNAs and their ability to form a trimeric complex with PABP, and to repress mRNA translation. UV crosslinking studies suggest that the ARS is less efficient than a poly(A) RNA of similar length, to bind to PABP. We show here that the ARS cannot efficiently form a trimeric complex with PABP; therefore, additional interactions with IMP1 and UNR to form a heteromeric RNP complex may be required for maximal repression of PABP mRNA translation under physiological conditions

Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro.

BACKGROUND: Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro. METHODS: To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin and the polycation transduction enhancer Transfectam. The EGFP-positive transduced cells were then enriched by flow cytometry. RESULTS: More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes. CONCLUSION: The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients own skeletal myoblasts

Fracture mechanics testing for environmental stress cracking in thermoplastics

Under the combined influence of an aggressive environment and applied stress, engineering thermoplastics may undergo a phenomenon known as environmental stress cracking (ESC). This can result in adverse effects such as embrittlement and premature failure in service, due to the growth of environmentally-induced cracks to critical sizes, with little to no fluid absorption in the bulk material. Fracture mechanics is proposed as a suitable scheme to study and quantify ESC, with the aim being to obtain characterising data for different polymer-fluid combinations of interest, as well as to develop a reliable fracture mechanics test protocol. In the proposed method, slow crack growth is monitored to assess the effect of a range of applied crack driving forces (K, or alternatively G) on observed crack speeds, as opposed to simply measuring time-to-failure. This paper presents the results of experiments performed on the following materials: linear low density polyethylene (LLDPE) in Igepal solution and high impact polystyrene (HIPS) in sunflower oil. A discussion of the various issues surrounding the data analysis for these long-term tests is also included, as the attainment of consistent and repeatable results is critical for a method to be internationally standardised, which is a goal of the European Structural Integrity Society (ESIS) Technical Committee 4 from whose interest this work is drawn

In silico and in vivo investigations using an endocavitary photoplethysmography sensor for tissue viability monitoring

Significance: Colorectal cancer is one of the major causes of cancer-related deaths worldwide. Surgical removal of the cancerous growth is the primary treatment for this disease. A colorectal cancer surgery, however, is often unsuccessful due to the anastomotic failure that may occur following the surgical incision. Prevention of an anastomotic failure requires continuous monitoring of intestinal tissue viability during and after colorectal surgery. To date, no clinical technology exists for the dynamic and continuous monitoring of the intestinal perfusion. Aim: A dual-wavelength indwelling bowel photoplethysmography (PPG) sensor for the continuous monitoring of intestinal viability was proposed and characterized through a set of in silico and in vivo investigations. Approach: The in silico investigation was based on a Monte Carlo model that was executed to quantify the variables such as penetration depth and detected intensity with respect to the sensor–tissue separations and tissue perfusion. Utilizing the simulated information, an indwelling reflectance PPG sensor was designed and tested on 20 healthy volunteers. Two sets of in vivo studies were performed using the driving current intensities 20 and 40 mA for a comparative analysis, using buccal tissue as a proxy tissue-site. Results: Both simulated and experimental results showed the efficacy of the sensor to acquire good signals through the “contact” to a “noncontact” separation of 5 mm. A very slow wavelength-dependent variation was shown in the detected intensity at the normal and hypoxic states of the tissue, whereas a decay in the intensity was found with the increasing submucosal-blood volume. The simulated detected-to-incident-photon-ratio and the experimental signal-to-noise ratio exhibited strong positive correlations, with the Pearson product-moment correlation coefficient R ranging between 0.65 and 0.87. Conclusions: The detailed feasibility analysis presented will lead to clinical trials utilizing the proposed sensor

BORIS/CTCFL is an RNA-binding protein that associates with polysomes

© 2013 Ogunkolade et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

Neuromedin U partially mediates leptin-induced hypothalamo-pituitary adrenal (HPA) stimulation and has a physiological role in the regulation of the HPA axis in the rat.

Intracerebroventricular (ICV) administration of the hypothalamic neuropeptide neuromedin U (NMU) or the adipostat hormone leptin increases plasma ACTH and corticosterone. The relationship between leptin and NMU in the regulation of the hypothalamo-pituitary adrenal (HPA) axis is currently unknown. In this study, leptin (1 nM) significantly increased the release of CRH from ex vivo hypothalamic explants by 207 ± 8.4% (P < 0.05 vs. basal), an effect blocked by the administration of anti-NMU IgG. The ICV administration of leptin (10 μg, 0.625 nmol) increased plasma ACTH and corticosterone 20 min after injection [plasma ACTH (picograms per milliliter): vehicle, 63 ± 20, leptin, 135 ± 36, P < 0.05; plasma corticosterone (nanograms per milliliter): vehicle, 285 ± 39, leptin, 452 ± 44, P < 0.01]. These effects were partially attenuated by the prior administration of anti-NMU IgG. Peripheral leptin also stimulated ACTH release, an effect attenuated by prior ICV administration of anti-NMU IgG. We examined the diurnal pattern of hypothalamic NMU mRNA expression and peptide content, plasma leptin, and plasma corticosterone. The diurnal changes in hypothalamic NMU mRNA expression were positively correlated with hypothalamic NMU peptide content, plasma corticosterone, and plasma leptin. The ICV administration of anti-NMU IgG significantly attenuated the dark phase rise in corticosterone [corticosterone (nanograms per milliliter): vehicle, 493 ± 38; NMU IgG, 342 ± 47 (P < 0.05)]. These studies suggest that NMU may play a role in the regulation of the HPA axis and partially mediate leptin-induced HPA stimulation. Copyright © 2006 by The Endocrine Society