Complexity of agonist- and cyclic AMP-mediated downregulation of the human beta 1-adrenergic receptor: role of internalization,degradation, and mRNA destabilization

Prolonged agonist exposure often induces downregulation of G protein-coupled receptors (GPCRs). Although downregulation of the prototypical beta(2)-adrenergic receptor (beta(2)AR) has been extensively studied, the underlying mechanisms have yet to be resolved. As even less is known about the beta(1)-subtype, we investigated the downregulation of human beta(1)AR stably expressed in Chinese hamster fibroblasts in response to the agonist isoproterenol or the cell-permeable, chlorophenylthio-cAMP (CPT-cAMP). While either effector mediated decreases in both beta(1)AR binding activity and steady-state beta(1)AR mRNA levels, there were significant differences in their actions. Whereas agonist-mediated downregulation of beta(1)AR followed first-order kinetics, that induced by CPT-cAMP was delayed for several hours and approximately 50% of the former. Furthermore, agonist but not CPT-cAMP induced beta(1)AR internalization, and inhibiting internalization also suppressed agonist-mediated downregulation. The latter, however, was more sensitive than the former to agonist concentration (EC(50) of 0.3 vs 48 nM). Thus, at < or =1 nM agonist, downregulation occurred without internalization and with a pattern similar to that mediated by CPT-cAMP. The amounts of beta(1)AR downregulated or internalized were proportional to initial receptor levels but reached saturation at approximately 2 and 3 pmol/mg of protein, respectively. The fate of beta(1)AR protein during downregulation was determined by immunoblotting with anti-C-terminal antibodies. In agonist-treated cells, beta(1)AR protein disappeared with time and without any immunoreactive degradation products. Agonist-mediated downregulation of the human beta(1)AR appears to be a complex process that consists of both agonist- and cAMP-specific components. The former involves both receptor internalization and degradation whereas the latter involves a reduction in receptor mRNA.     

HPLC-MS/MS analysis of the products generated from all-trans-retinoic acid using recombinant human CYP26A

Two mammalian hCYP26A expression systems have been used to analyze the metabolic products of CYP26A. Through the use of extensive HPLC, UV spectroscopy, and liquid chromatography/tandem mass spectrometry (LC-MS/MS) methodology, we have conclusively demonstrated that the complex mixture of products comprises 4-OH-all-trans-retinoic acid, 4-oxo-all-trans-retinoic acid, and 18-OH-all-trans-retinoic acid, and more polar products, partially identified as dihydroxy and mono-oxo, mono-hydroxy derivatives. These more polar products are presumed to result from multiple hydroxylations on the beta-ionone ring. The inter-relationship of initial and polar metabolites was inferred from both gene-dose and time-course experiments. Both initial and secondary metabolic steps after 4-oxo-all-trans-retinoic acid are ketoconazole-sensitive, suggesting that steps in the production of water-soluble metabolites are cytochrome P450-dependent.     

Temporal genetic variation in Aedes aegypti populations in Ho Chi Minh City (Vietnam)

Aedes aegypti, the main vector of dengue viruses in Asia, displays variation in population density over time. The larval habitats of this species being unevenly distributed and transient (depending on cycles of drought and flood), the forces generating temporal variation in gene frequencies in populations are studied. We sampled seven mosquito populations from Ho Chi Minh City (Vietnam) and its suburbs on five occasions between April 1999 and August 2000. We investigated genetic variation by studying isoenzyme and microsatellite polymorphism and susceptibility to a dengue 2 virus strain. Ae. aegypti populations collected during the dry season (January-April) showed genetic differentiation (F(ST) = 0.016, P < 10(-6) for isoenzymes) and showed more differentiated infection rates of the dengue 2 virus. The genetic structure of the population is less marked during the rainy season (F(ST) = 0.081, P < 10(-6)). Thus, environmental factors, such as rainfall and factors related to human activity, such as breeding site density and insecticide treatment, control the genetic structure of Ae. aegypti populations in the short term. The implications of studies of this kind for the design of future control programmes are discussed.     

Thelephantins D-H: five p-terphenyl derivatives from the inedible mushroom Thelephora aurantiotincta

Five p-terphenyl derivatives named thelephantins D-H (1-5) together with nine known compounds, thelephantins A-C (6-8), ganbajunin E (9), p-hydroxylbenzoic acid (10), ganbajunin C (11), thelephorin A (12), 2-O-methylatromentin (13) and atromentin (14), were isolated from the methanolic extract of fruit bodies of the Thelephoraceous Basidiomycete Thelephora aurantiotincta. Their structures were elucidated by high-resolution MS, 2D NMR, IR and UV spectroscopic analysis.     

Use of polyethylenimine-adenovirus complexes to examine triplex formation in intact cells

Triplex-forming oligonucleotides (TFOs) show potential for sequence-specific DNA binding and inhibition of gene expression. We have applied this antigene strategy using a TFO incorporating an Auger-emitting radionucleotide, 125I, to study the production of double-strand breaks (dsb) in the rat aquaporin 5 (rAQP5) cDNA. 125I-TFO bound to the pCMVrAQP5 plasmid in vitro in a dose-dependent manner and formed stable triplexes up to 65 degrees C and in the presence of 140 mM KCl. Further, 125I-TFO resulted in a predictable dsb when analyzed by Southern hybridization. To deliver TFOs to epithelial cells, we employed 125I-TFO-polyethyleneimine-adenovirus (125I-TFO-PEI-Ad) complexes. We hypothesized that these complexes would take advantage of adenoviral characteristics to transfer 125I-TFO to the cell nucleus. Adenovirus-containing complexes brought about greater uptake and nuclear localization of TFOs compared with delivery with 125I-TFO-PEI complexes alone. No significant degradation of 125I-TFO was found after delivery into cells using PEI-Ad complexes and freezing and thawing. We next used PEI-Ad complexes to deliver 125I-TFO and pCMVrAQP5 separately to epithelial cells to determine if triplexes can form de novo within cells, resulting in the specific dsb in the rAQP5 cDNA. After delivery, cell pellets were stored at -80 degrees C for more than 60 days. Thereafter, plasmid DNA was isolated from cells and analyzed for dsb by Southern hybridization. However, none were detected. We conclude that under the experimental conditions employed, effective triplexes, with 125I-TFO and pCMVrAQP5, do not form de novo inside cells.     

A new rhodamine‐based fluorescent chemodosimeter for mercuric ions in water media

A new rhodamine–ethylenediamine–nitrothiourea conjugate (RT) was synthesized and its sensing property as a fluorescent chemodosimeter toward metal ions was explored in water media. Analytical results from absorption and fluorescence spectra revealed that the addition of Hg²⁺ ions to the aqueous solution of the chemodosimeter RT caused a distinct fluorescence OFF–ON response with a remarkable visual color change from colorless to pink; however, no clear spectral and color changes were observed from other metal ions including: Zn²⁺, Cu²⁺, Cd²⁺, Pb²⁺, Ag⁺, Fe²⁺, Cr³⁺, Co³⁺, Ni²⁺, Ca²⁺, Mg²⁺, K⁺ and Na⁺. The sensing results and the molecular structure suggested that a Hg²⁺‐induced a desulfurization reaction and cyclic guanylation of the thiourea moiety followed by ring‐opening of the rhodamine spirolactam in RT are responsible for a distinct fluorescence turn‐on signal, indicating that RT is a remarkably sensitive and selective chemodosimeter for Hg²⁺ ions in aqueous solution. Hg²⁺ within a concentration range from 0.1 to 25 μM can be determined using RT as a chemodosimeter and a detection limit of 0.04 μM is achieved. Copyright © 2014 John Wiley & Sons, Ltd.     

Time-Dependent Increase in Network Response to Stimulation

In vitro neuronal cultures have become a popular method with which to probe network-level neuronal dynamics and phenomena in controlled laboratory settings. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Here we demonstrate the effects of a high frequency electrical stimulation signal in training cultured networks of cortical neurons. Networks receiving this training signal displayed a time-dependent increase in the response to a low frequency probing stimulation, particularly in the time window of 20–50 ms after stimulation. This increase was found to be statistically significant as compared to control networks that did not receive training. The timing of this increase suggests potentiation of synaptic mechanisms. To further investigate this possibility, we leveraged the powerful Cox statistical connectivity method as previously investigated by our group. This method was used to identify and track changes in network connectivity strength.     

Physical Activity in Vietnam: Estimates and Measurement Issues

IntroductionOur aims were to provide the first national estimates of physical activity (PA) for Vietnam, and to investigate issues affecting their accuracy.MethodsMeasurements were made using the Global Physical Activity Questionnaire (GPAQ) on a nationally-representative sample of 14706 participants (46.5% males, response 64.1%) aged 25−64 years selected by multi-stage stratified cluster sampling.ResultsApproximately 20% of Vietnamese people had no measureable PA during a typical week, but 72.9% (men) and 69.1% (women) met WHO recommendations for PA by adults for their age. On average, 52.0 (men) and 28.0 (women) Metabolic Equivalent Task (MET)-hours/week (largely from work activities) were reported. Work and total PA were higher in rural areas and varied by season. Less than 2% of respondents provided incomplete information, but an additional one-in-six provided unrealistically high values of PA. Those responsible for reporting errors included persons from rural areas and all those with unstable work patterns. Box-Cox transformation (with an appropriate constant added) was the most successful method of reducing the influence of large values, but energy-scaled values were most strongly associated with pathophysiological outcomes.ConclusionsAround seven-in-ten Vietnamese people aged 25–64 years met WHO recommendations for total PA, which was mainly from work activities and higher in rural areas. Nearly all respondents were able to report their activity using the GPAQ, but with some exaggerated values and seasonal variation in reporting. Data transformation provided plausible summary values, but energy-scaling fared best in association analyses.     

Sense-overlapping lncRNA as a decoy of translational repressor protein for dimorphic gene expression

Long noncoding RNAs (lncRNAs) are vastly transcribed and extensively studied but lncRNAs overlapping with the sense orientation of mRNA have been poorly studied. We analyzed the lncRNA DAPALR overlapping with the 5´ UTR of the Doublesex1 (Dsx1), the male determining gene in Daphnia magna. By affinity purification, we identified an RNA binding protein, Shep as a DAPALR binding protein. Shep also binds to Dsx1 5´ UTR by recognizing the overlapping sequence and suppresses translation of the mRNA. In vitro and in vivo analyses indicated that DAPALR increased Dsx1 translation efficiency by sequestration of Shep. This regulation was impaired when the Shep binding site in DAPALR was deleted. These results suggest that Shep suppresses the unintentional translation of Dsx1 by setting a threshold; and when the sense lncRNA DAPALR is expressed, DAPALR cancels the suppression caused by Shep. This mechanism may be important to show dimorphic gene expressions such as sex determination and it may account for the binary expression in various developmental processes.