The mechanism of recovery of hepatic T4-5'-deiodinase during glucose-refeeding: role of glucagon and insulin

Refeeding studies were performed on male Sprague-Dawley rats that had been fasted for 72 hours to characterize the specific effect of carbohydrates on T₃ metabolism. Fasting is associated with low serum T₃ levels and reduced hepatic T₄-5′-deiodinase activity (T₄ → T₃). Carbohydrate refeeding (20% glucose in H₂O) normalized both the serum T₃ and hepatic T₄-5′-deiodinase activity within 72 hours, whereas fat (10% Intralipid) and amino acids (5.5% Travasol) had no effect after 72 hours of refeeding. Refeeding with a mixed diet (Purina Rodent Chow) occasionally reactivated hepatic T₄-5′-deiodinase activity, however, normalization of enzyme activity did not occur within 72 hours. Time-course studies demonstrated that hepatic T₄-5′-deiodinase activity was not stimulated until 24 hours of carbohydrate refeeding had elapsed and that 48 to 72 hours were required for normalization. The mechanism of the carbohydrate-refeeding effect was characterized by analyzing the alterations in the kinetics Michaelis constant (Km) and maximal velocity (Vmax) of hepatic T₄-5′-deiodinase and the changes in the hepatic content of nonprotein sulfhydryl groups (NP-SH), which are possible enzyme cofactors. There was no relationship between the hepatic enzyme activity and the NP-SH response during the refeeding period. Moreover, homogenate enrichment with the sulfhydryl compound, dithiothreitol (DTT), did not alter the temporal profile of the enzyme recovery consequent to refeeding. Refeeding with carbohydrate had no effect on the Km of hepatic T₄-5′-deiodinase but had a significant effect on the Vmax. Refeeding with glucose induced an increase in enzyme Vmax over the time-course, which became significant (P < 0.005) compared with the enzyme Vmax of the fasted group by 72 hours. During carbohydrate refeeding, a positive correlation was noted between the ratio of serum insulin to glucagon and hepatic-T₄-5′-deiodinase activity (r = 0.82, P < 0.001), whereas a negative correlation was found between enzyme activity and the ratio of serum glucose to insulin (r = ?0.9, P < 0.001). Furthermore these correlations also applied during refeeding with fat and amino acids. Thus, the carbohydrate-refeeding reactivation of hepatic T₄-5′-deiodinase in fasted rats is a delayed process that requires a refeeding period equivalent to the duration of fasting for enzyme normalization to occur. Recovery was due to an increase in the hepatic content of active enzyme rather than an enhancement of cofactor supply. The glucoregulatory hormones, glucagon and insulin, may modulate these carbohydrate induced changes on hepatic T₄-5′-deiodinase. Moreover, the differential reaction of hepatic T₄-5′-deiodinase to specific nutriments may be mediated by these glucoregulatory hormones.     

Sequence Conservation of the Region Targeted by the Abbott RealTime HBV Viral Load Assay in Clinical Specimens

The Abbott RealTime HBV assay targets the N-terminal region of the S gene. Here we analyzed the sequence variability of the assay target region from >2,100 clinical specimens. Thermodynamic modeling of the percentage of bound primer/probe at the assay annealing temperature was performed to assess the potential effect of sequence variability.     

Comparison of two commercial ELISA systems for evaluating anti‐EBNA1 IgG titers

High IgG titers against the Epstein–Barr virus nuclear antigen, EBNA‐1, have been strongly correlated with the risk of developing multiple sclerosis. ELISAs are used frequently to measure EBNA‐1 titers, however concerns remain regarding the accuracy of results. Ordering absolute results into rank quintiles for analysis may be preferable. Using 120 serum samples, two commercially available ELISAs (produced by DiaSorin and VirionSerion) were compared, both in terms of absolute results and rank quintiles. The positive predictive value of the VirionSerion ELISA was 99.1% when compared to the DiaSorin ELISA, however, the negative predictive value was 64.3%. Sensitivity and specificity were acceptable at 95.5% and 90.0%, respectively. There was poor correlation between absolute results, R² = 0.49; and the kappa coefficient for rank quintiles was low at 0.23. Although sensitivity and specificity appear adequate, the poor negative predictive value and kappa coefficient are of major concern. Care must be taken when selecting assays for experimental use. J. Med. Virol. 85:128–131, 2012. © 2012 Wiley Periodicals, Inc.     

Effects of short-term exercise-training on aortic systolic pressure augmentation in overweight and obese individuals

To determine whether exercise training-induced decreases in blood pressure (BP) can be explained by decreases in aortic systolic pressure augmentation in overweight or obese individuals. Thirty-five sedentary or recreationally active men and women (30–57 years) who were either overweight (40 %) or obese (60 %) completed 6 weeks of exercise training (≥3 days/week; stationary bike and/or treadmill) either preceded (n = 19) or followed (n = 16) by a 6-week control period of no exercise. Aortic augmentation pressure (AP), aortic and peripheral augmentation indices (AIx), and central aortic BP (SphygmoCor) were determined before and after exercise training and a control period. Peak oxygen consumption increased (p = 0.0001) from 27.0 ± 5.1 to 28.8 ± 5.8 mL/(kg min) after 6 weeks of exercise. Exercise training decreased brachial systolic (SBP) and diastolic BP from 142 ± 8/94 ± 8 to 134 ± 11/86 ± 11 mmHg (p < 0.005/p < 0.005); whereas no changes were observed after the control period (141 ± 11/91 ± 9 mmHg, p = 0.81/p = 0.34). Neither AP (baseline: 9.2 ± 4.2 mmHg; after 6 weeks training: 8.7 ± 6.1 mmHg), aortic AIx (baseline: 24.6 ± 11.0 %; after 6 weeks training: 22.7 ± 11.1 %), nor peripheral AIx (baseline: 81.4 ± 16.7 mmHg; after 6 weeks training: 76.4 ± 16.5 mmHg) were modified by exercise training. Although aortic SBP decreased after exercise (132 ± 8 to 124 ± 12 mmHg, p < 0.002), these changes were accounted for by decreases in mean arterial pressure. In overweight or obese individuals, although short-term aerobic exercise training, which improved cardiorespiratory fitness, may produce marked decreases in aortic and brachial BP; these effects are not attributed to alterations in aortic systolic pressure augmentation.     

Synthesis and biochemical evaluation of cephalosporin analogues equipped with chemical tethers

Molecular probes typically require structural modifications to allow for the immobilisation or bioconjugation with a desired substrate but the effects of these changes are often not evaluated. Here, we set out to determine the effects of attaching functional handles to a first-generation cephalosporin. A series of cephalexin derivatives was prepared, equipped with chemical tethers suitable for the site-selective conjugation of antibiotics to functionalised surfaces. The tethers were positioned remotely from the β-lactam ring to ensure minimal effect to the antibiotic''s pharmacophore. Herein, the activity of the modified antibiotics was evaluated for binding to the therapeutic target, the penicillin binding proteins, and shown to maintain binding interactions. In addition, the deactivation of the modified drugs by four β-lactamases (TEM-1, CTX-M-15, AmpC, NDM-1) was investigated and the effect of the tethers on the catalytic efficiencies determined. CTX-M-15 was found to favour hydrolysis of the parent antibiotic without a tether, whereas AmpC and NDM-1 were found to favour the modified analogues. Furthermore, the antimicrobial activity of the derivatives was evaluated to investigate the effect of the structural modifications on the antimicrobial activity of the parent drug, cephalexin.

Tethered β-lactam antibiotics provide insights into designing chemical tools to target specific β-lactamases.     

Isolation and characterisation of microsatellite loci for the ancient cephalopod,Nautilus pompilius

Conservation Genetics Resources - A microsatellite-enriched genomic library was created from a single Nautilus pompilius individual and clones/fragments sequenced using Sanger and Illumina MiSeq...     

Profiling receptor tyrosine kinase activation by using Ab microarrays

Signal transduction in mammalian cells is mediated by complex networks of interacting proteins. Understanding these networks at a circuit level requires devices to measure the amounts and activities of multiple proteins in a rapid and accurate manner. Ab microarrays have previously been applied to the quantification of labeled recombinant proteins and proteins in serum. The development of methods to analyze intracellular signaling molecules on microarrays would make Ab arrays widely useful in systems biology. Here we describe the fabrication of multiplex Ab arrays sensitive to the amounts and modification states of signal transduction proteins in crude cell lysates and the integration of these arrays with 96-well microtiter plate technology to create microarrays in microplates. We apply the Ab arrays to monitoring the activation, uptake, and signaling of ErbB receptor tyrosine kinases in human tumor cell lines. Data obtained from multicolor ratiometric microarrays correlate well with data obtained by using traditional approaches, but the arrays are faster and simpler to use. The integration of microplate and microarray methods for crude cell lysates should make it possible to identify and analyze small molecule inhibitors of signal transduction processes with unprecedented speed and precision. We demonstrate the future potential of this approach by characterizing the action of the epidermal growth factor receptor inhibitor PD153035 on cells by using Ab arrays; direct scale-up to array-based screening in 96- and 384-well plates should allow small molecules to be identified with specific inhibitory profiles against a signaling network.