Thyroid hormone-regulated enhancer blocking: cooperation of CTCF and thyroid hormone receptor

The highly conserved, ubiquitously expressed, zinc finger protein CTCF is involved in enhancer blocking, a mechanism crucial for shielding genes from illegitimate enhancer effects. Interestingly, CTCF-binding sites are often flanked by thyroid hormone response elements (TREs), as at the chicken lysozyme upstream silencer. Here we identify a similar composite site positioned upstream of the human c-myc gene. For both elements, we demonstrate that thyroid hormone abrogates enhancer blocking. Relief of enhancer blocking occurs even though CTCF remains bound to the lysozyme chromatin. Furthermore, chromatin immunoprecipitation analysis of the lysozyme upstream region revealed that histone H4 is acetylated at the CTCF-binding site. Loss of enhancer blocking by the addition of T3 led to increased histone acetylation, not only at the CTCF site, but also at the enhancer and the promoter. Thus, when TREs are adjacent to CTCF-binding sites, thyroid hormone can regulate enhancer blocking, thereby providing a new property for what was previously thought to be constitutive enhancer shielding by CTCF.     

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells

The time course of incorporation of [¹⁴C]arachidonic acid and [³H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [¹⁴C]arachidonic acid and [³H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

Unusual redox behaviour of cytochrome b-561 from bovine chromaffin granule membranes

(1) Redox titrations of cytochrome b-561 have been performed with the purified cytochrome and with intact and detergent-solubilized chromaffin-granule membranes. (2) The midpoint redox potential of the cytochrome is 100–130 mV; this depends upon the composition of the buffer, but is independent of pH in the range 5.5–7.5; partial proteolysis of the cytochrome raises the midpoint potential to 160 mV. (3) The Nernst plots of titration data have slopes of 75–115 mV, and are in some cases sigmoid in shape. This may be explained by negative cooperativity during redox transitions in oligomeric cytochrome b-561. (4) Measurements of the haem and cytochrome content of chromaffin granule membrane suggest a haem content of 1 mol/mol protein. (5) Chemical crosslinking of cytochrome b-561 suggests that it may exist as an oligomer of 4–6 polypeptide chains within the chromaffin granule membrane. Aggregation of purified cytochrome b-561 was shown by gel filtration studies and by immunological methods in SDS-polyacrylamide gels. Studies of the molecular weight of the aggregates suggest that the monomer has a molecular weight close to 22 000, but migrates anomalously slowly during electrophoresis.     

Design, synthesis and structure-activity relationships of (indo-3-yl) heterocyclic derivatives as agonists of the CB1 receptor. Discovery of a clinical candidate

We report an expansion of the structure-activity relationship (SAR) of a novel series of indole-3-heterocyclic CB1 receptor agonists. Starting from the potent but poorly soluble lead, 1, a rational approach was taken in order to balance solubility, hERG activity and potency while retaining the desired long duration of action within the mouse tail flick test. This led to the discovery of compound 38 which successfully progressed into clinical development.     

Trends in rates of different forms of diagnosed coronary heart disease, 1978 to 2000: prospective,population based study of British men

Objective To examine trends over time in rates of different forms of diagnosed coronary heart disease among British men, during a period in which mortality due to coronary heart disease has been declining.Design Prospective cohort study covering the period 1978-80 to 1998-2000.Participants 7735 men, aged 40-59 at entry, randomly selected from one general practice in each of 24 British towns.Main outcome measures Trends in the rates of major coronary events, first diagnosed angina and first diagnosed coronary heart disease (any fatal or non-fatal documented event or diagnosis). Events were ascertained from NHS central registers and reviews of medical records from general practices.Results Over the 20 year period, 1561 major coronary events occurred; 1087 and 1816 men had new diagnoses of angina and coronary heart disease, respectively. The age adjusted annual relative changes were -3.6% (95% confidence interval -4.8% to -2.4%, P < 0.001) for all major coronary events, 2.6% (1.1% to 4.0%, P < 0.001) for first diagnosed angina and -0.8% (-1.8% to 0.3%, P = 0.18) for first diagnosed coronary heart disease. The fall in major coronary events occurred across all categories of event (fatal and non-fatal, first and recurrent). Similarly, first diagnosed angina increased for both uncomplicated angina and angina after myocardial infarction. The age adjusted annual relative change in case fatality at 28 days of first major coronary events was -1.4% (-3.1% to 0.4%, P = 0.12).Conclusions Among British middle aged men, a substantial decline in the rate of major coronary events over the past two decades seems to have been largely offset by an increase in the incidence of diagnosed angina. Overall there was little change in the incidence of first diagnosed coronary heart disease. A continuing need exists for resources and services for coronary heart disease in general, and for new angina in particular.     

The basement membrane of hair follicle stem cells is a muscle cell niche

The hair follicle bulge in the epidermis associates with the arrector pili muscle (APM) that is responsible for piloerection ("goosebumps"). We show that stem cells in the bulge deposit nephronectin into the underlying basement membrane, thus regulating the adhesion of mesenchymal cells expressing the nephronectin receptor, α8β1 integrin, to the bulge. Nephronectin induces α8 integrin-positive mesenchymal cells to upregulate smooth muscle markers. In nephronectin knockout mice, fewer arrector pili muscles form in the skin, and they attach to the follicle above the bulge, where there is compensatory upregulation of the nephronectin family member EGFL6. Deletion of α8 integrin also abolishes selective APM anchorage to the bulge. Nephronectin is a Wnt target; epidermal β-catenin activation upregulates epidermal nephronectin and dermal α8 integrin expression. Thus, bulge stem cells, via nephronectin expression, create a smooth muscle cell niche and act as tendon cells for the APM. Our results reveal a functional role for basement membrane heterogeneity in tissue patterning. PAPERCLIP:     

Reliability of surface EMG matrix in locating the innervation zone of upper trapezius muscle

The identification of the motor unit (MU) innervation zone (IZ) using surface electromyographic (sEMG) signals detected on the skin with a linear array or a matrix of electrodes has been recently proposed in the literature. However, an analysis of the reliability of this procedure and, therefore, of the suitability of the sEMG signals for this purpose has not been reported.The purpose of this work is to describe the intra and inter-rater reliability and the suitability of surface EMG in locating the innervation zone of the upper trapezius muscle.Two operators were trained on electrode matrix positioning and sEMG signal analysis. Ten healthy subjects, instructed to perform a series of isometric contractions of the upper trapezius muscle participated in the study. The two operators collected sEMG signals and then independently estimated the IZ location through visual analysis.Results showed an almost perfect agreement for intra-rater and inter-rater reliability. The constancy of IZ location could be affected by the factors reflecting the population of active MUs and their IZs, including: the contraction intensity, the acquisition period analyzed, the contraction repetition. In almost all cases the IZ location shift due to these factors did not exceed 4 mm. Results generalization to other muscles should be made with caution.     

Differential regulation of the ascorbic acid transporter SVCT2 during development and in response to ascorbic acid depletion

The sodium-dependent vitamin C transporter-2 (SVCT2) is the only ascorbic acid (ASC) transporter significantly expressed in brain. It is required for life and is critical during brain development to supply adequate levels of ASC. To assess SVCT2 function in the developing brain, we studied time-dependent SVCT2 mRNA and protein expression in mouse brain, using liver as a comparison tissue because it is the site of ASC synthesis. We found that SVCT2 expression followed an inverse relationship with ASC levels in the developing brain. In cortex and cerebellum, ASC levels were high throughout late embryonic stages and early post-natal stages and decreased with age, whereas SVCT2 mRNA and protein levels were low in embryos and increased with age. A different response was observed for liver, in which ASC levels and SVCT2 expression were both low throughout embryogenesis and increased post-natally. To determine whether low intracellular ASC might be capable of driving SVCT2 expression, we depleted ASC by diet in adult mice unable to synthesize ASC. We observed that SVCT2 mRNA and protein were not affected by ASC depletion in brain cortex, but SVCT2 protein expression was increased by ASC depletion in the cerebellum and liver. The results suggest that expression of the SVCT2 is differentially regulated during embryonic development and in adulthood.     

AMPK: a nutrient and energy sensor that maintains energy homeostasis

AMP-activated protein kinase (AMPK) is a crucial cellular energy sensor. Once activated by falling energy status, it promotes ATP production by increasing the activity or expression of proteins involved in catabolism while conserving ATP by switching off biosynthetic pathways. AMPK also regulates metabolic energy balance at the whole-body level. For example, it mediates the effects of agents acting on the hypothalamus that promote feeding and entrains circadian rhythms of metabolism and feeding behaviour. Finally, recent studies reveal that AMPK conserves ATP levels through the regulation of processes other than metabolism, such as the cell cycle and neuronal membrane excitability.