A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Upregulation of Ca2+ removal in human skeletal muscle: a possible role for Ca2+-dependent priming of mitochondrial ATP synthesis

In muscle, ATP is required for the powerstroke of the myosin head, the detachment of actin and myosin filaments, and the reuptake of Ca²⁺ into the sarcoplasmic reticulum. During contraction-relaxation, large amounts of ATP are consumed at the sites of action of the myosin-ATPase and sarcoplasmic reticulum Ca²⁺-ATPase. The present study addresses the consequences of a reduction in mitochondrial ATP production capacity on sarcoplasmic Ca²⁺ handling. To this end, myotubes were cultured from patient quadriceps with a biochemically defined decrease in the maximal rate of mitochondrial ATP production and were loaded with indo 1 for imaging of sarcoplasmic Ca²⁺ changes in real time by confocal microscopy. Myotubes were field-stimulated with 10-ms pulses of 16 V to evoke transient rises in sarcoplasmic Ca²⁺ concentration ([Ca²⁺]_S). Three single pulses, two pulse trains (1 Hz), and one single pulse were applied in succession to mimic changing workloads. Control myotubes displayed [Ca²⁺]_S transients with an amplitude that was independent of the strength of the stimulus. Intriguingly, the rate of sarcoplasmic Ca²⁺ removal (CRR) was significantly upregulated during the second and subsequent transients. In myotubes with a reduced mitochondrial ATP production capacity, the amplitude of the [Ca²⁺]_S transients was markedly increased at higher stimulus intensities. Moreover, upregulation of the CRR was significantly decreased compared with control. Taken together, these results are in good agreement with a tight coupling between mitochondrial ATP production and sarcoplasmic Ca²⁺ handling. Moreover, they support the existence of a relatively long-lasting mitochondrial memory for sarcoplasmic [Ca²⁺] rises. This memory, which manifested itself as an increase in CRR upon recurrent stimulation, was impaired in patient myotubes with a reduced mitochondrial ATP production capacity. sarcoplasmic Ca²⁺ removal; video-rate imaging; indo 1; electrical stimulation; mitochondrial memory     

The Terneuzen Birth Cohort: BMI Changes between 2 and 6 Years Correlate Strongest with Adult Overweight

Background

Complications of overweight amplify with age, and irreversible damage already exists in young persons. Identifying the most sensitive age interval(s) for adult overweight is relevant for primary prevention. The aim of the study was to assess the relative contribution of body mass index (BMI) changes between 0 and 18 years to adult overweight, and to identify the earliest critical growth period.

Methods and Findings

Data from 762 subjects in the Terneuzen Birth Cohort with an average of 21 growth measurements per subject from birth until 18 years were used. The main outcome measure was the BMI standard deviation score (SDS) at young adulthood. For each subject BMI SDS was fitted by a piecewise linear model at eight different ages and correlated to adult BMI SDS. The age intervals in between are considered critical according to three criteria, tested by respectively Students'' t-tests, multiple linear regression analyses and Pearson''s correlation tests. In the age intervals 4 months(m) -1 year(y), 2–6 y, 6–10 y and 10–18 y the BMI SDS change differs between adults with and without overweight (P≤0.001). The age intervals 2–6 y and 10–18 y also meet the second criterion, implying that the BMI change during this period has a predictive value for adult BMI SDS in addition to BMI SDS at the end of the period. The largest rise in correlation between estimated BMI SDS and measured adult BMI SDS occurs during the period 2–6 y (from 0.36 to 0.63), which results in a high sensitivity (0.6) and specificity (0.8) by the age of 6 y.

Conclusions/Significance

The age interval from 2 y to 6 y is the earliest and most critical growth period for adult overweight. Therefore, primary prevention of adult overweight seems most likely to be successful if targeted at this specific age interval. By identifying those with an upwards centile crossing between 2 and 6 years, the development towards adult overweight might be reversed.     

Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2′-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.     

Baseline cytosolic Ca2+ oscillations derived from a non-endoplasmic reticulum Ca2+ store

Cytosolic Ca(2+) oscillations can be due to cycles of release and re-uptake of internally stored Ca(2+). To investigate the nature of these Ca(2+) stores, we expressed the Pmr1 Ca(2+) pump of Caenorhabditis elegans in COS-1 cells and pretreated the cells with thapsigargin to prevent Ca(2+) uptake by the sarco(endo)plasmic reticulum Ca(2+)-ATPase. Pmr1 co-localized with the Golgi-specific 58K protein and was targeted to a Ca(2+) store that was less leaky for Ca(2+) than the endoplasmic reticulum and whose inositol trisphosphate receptors were less sensitive to inositol trisphosphate and ATP than those in the endoplasmic reticulum. ATP-stimulated Pmr1-overexpressing cells responded after a latency to extracellular Ca(2+) with a regenerative Ca(2+) signal, which could be prevented by caffeine. They also produced very stable ilimaquinone-sensitive baseline Ca(2+) spikes, even in the presence of thapsigargin. Such responses never occurred in non-transfected cells or in cells that overexpressed the type-1 sarco(endo)plasmic reticulum Ca(2+)-ATPase. Abortive Ca(2+) spikes also occurred in histamine-stimulated untransfected HeLa cells pretreated with thapsigargin, and they too were inhibited by ilimaquinone. We conclude that the Pmr1-induced Ca(2+) store, which probably corresponds to the Golgi compartment, can play a crucial role in setting up baseline Ca(2+) spiking.     

TV Time but Not Computer Time Is Associated with Cardiometabolic Risk in Dutch Young Adults

Background

TV time and total sedentary time have been positively related to biomarkers of cardiometabolic risk in adults. We aim to examine the association of TV time and computer time separately with cardiometabolic biomarkers in young adults. Additionally, the mediating role of waist circumference (WC) is studied.

Methods and Findings

Data of 634 Dutch young adults (18–28 years; 39% male) were used. Cardiometabolic biomarkers included indicators of overweight, blood pressure, blood levels of fasting plasma insulin, cholesterol, glucose, triglycerides and a clustered cardiometabolic risk score. Linear regression analyses were used to assess the cross-sectional association of self-reported TV and computer time with cardiometabolic biomarkers, adjusting for demographic and lifestyle factors. Mediation by WC was checked using the product-of-coefficient method.TV time was significantly associated with triglycerides (B = 0.004; CI = [0.001;0.05]) and insulin (B = 0.10; CI = [0.01;0.20]). Computer time was not significantly associated with any of the cardiometabolic biomarkers. We found no evidence for WC to mediate the association of TV time or computer time with cardiometabolic biomarkers.

Conclusions

We found a significantly positive association of TV time with cardiometabolic biomarkers. In addition, we found no evidence for WC as a mediator of this association. Our findings suggest a need to distinguish between TV time and computer time within future guidelines for screen time.     

Comparison of conventional and molecular methods for the detection of bacterial pathogens in sputum samples from cystic fibrosis patients

The nature of the micro-flora present in sputa of six different cystic fibrosis (CF) patients was assessed using routine microbiological culture and molecular methods. Bacterial genes for the small subunit ribosomal RNA (ssu rDNA) were specifically amplified from DNA extracted from the sputum samples, cloned and characterised by hybridisation and DNA sequencing. A large number of clones from six sputa were screened. Initially, oligonucleotide hybridisation was performed with five probes, specific for Gram-positives and Gram-negatives in general and the main pathogens for the CF patient (Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae). For a single sputum sample, the results were fully congruent when culture and molecular methods were compared. In the other five sputa, discrepancies for S. aureus and/or H. influenzae were documented. Although S. aureus DNA and H. influenzae DNA was detected in three and four sputa, respectively, strains could not be cultured. Although the PCR approach is not capable of distinguishing viable from dead bacteria, all of the CF patients had a history of S. aureus infections, while one of the CF patients once had cultivable H. influenzae in the sputum as well. A number of clones for probe-unidentified Gram-negative or Gram-positive bacterial species were further analysed by sequencing and additional potential pathogens were identified. Although routine culture of sputum frequently points to mono-specific exacerbations, our molecular data indicate that the other CF-related pathogens appear to be persistently present as well. We conclude that routine culture for bacterial pathogens from CF sputa yields limited microbiological information since it frequently fails to identify a number of pathogenic bacterial species that are potentially present in a viable status in the lungs of these patients.