Higher order genomic organization and epigenetic control maintain cellular identity and prevent breast cancer.

Cells establish and sustain structural and functional integrity of the genome to support cellular identity and prevent malignant transformation. In this review, we present a strategic overview of epigenetic regulatory mechanisms including histone modifications and higher order chromatin organization (HCO) that are perturbed in breast cancer onset and progression. Implications for dysfunctions that occur in hormone regulation, cell cycle control, and mitotic bookmarking in breast cancer are considered, with an emphasis on epithelial-to-mesenchymal transition and cancer stem cell activities. The architectural organization of regulatory machinery is addressed within the contexts of translating cancer-compromised genomic organization to advances in breast cancer risk assessment, diagnosis, prognosis, and identification of novel therapeutic targets with high specificity and minimal off target effects.

Evidence required to demonstrate clinical utility of pharmacogenetic testing: the debate continues.

Pharmacogenetics is an area of research that has potential to greatly benefit patients. However, the routine use of diagnostic pharmacogenetic testing to inform treatment decisions is limited. Here we discuss the determination of clinical utility of pharmacogenetic testing and the level of evidence required to support translation into clinical practice.

Clinical implementation of germ line cancer pharmacogenetic variants during the next-generation sequencing era.

More than 100 medications approved by the US Food and Drug Administration include pharmacogenetic biomarkers in the drug label, many with cancer indications referencing germ line DNA variations. With the advent of next-generation sequencing (NGS) and its rapidly increasing uptake into cancer research and clinical practice, an enormous amount of data to inform documented gene-drug associations will be collected that must be exploited to optimize patient benefit. This review focuses on the implementation of germ line cancer pharmacogenetics in clinical practice. Specifically, it discusses the importance of germ line variation in cancer and the role of NGS in pharmacogenetic discovery and implementation. In the context of a scenario in which massive amounts of NGS-based genetic information will be increasingly available to health stakeholders, this review explores the ongoing debate regarding the threshold of evidence necessary for implementation, provides an overview of recommendations in cancer by professional organizations and regulatory bodies, and discusses limitations of current guidelines and strategies to improve third-party coverage.

On the inhibition of hepatic glycogenolysis by fructose. A 31P-NMR study in perfused rat liver using the fructose analogue 2,5-anhydro-D-mannitol.

Inhibition of hormone-stimulated hepatic glycogenolysis by fructose (Fru) has been attributed to accumulation of the competitive inhibitor Fru1P and/or to the associated depletion of the substrate phosphate (Pi). To evaluate the relative importance of either factor, we used the Fru analogue 2,5-anhydro-D-mannitol (aHMol). This analogue is avidly phosphorylated, traps Pi, and inhibits hormone-stimulated glycogenolysis, but it is not a gluconeogenic substrate, and hence does not confound glycogenolytic glucose production. Livers were continuously perfused with dibutyryl-cAMP (100 microM) to clamp phosphorylase in its fully activated a form. We administered aHMol (3.8 mM), and studied changes in glycogenolysis (glucose, lactate and pyruvate output) and in cytosolic Pi and phosphomonoester (PME), using in situ 31P-NMR spectroscopy (n = 4). Lobes of seven livers perfused outside the magnet were extracted for evaluation, by high-resolution 31P-NMR, of the evolution of aHMol1P and of aHMol(1,6)P2. After addition of aHMol, both glycogenolysis and the NMR Pi signal dropped precipitously, while the PME signal rose continuously and was almost entirely composed of aHMol1P. Inhibition of glycogenolysis in excess of the drop in Pi could be explained by continuing accumulation of aHMol1P. A subsequent block of mitochondrial ATP synthesis by KCN (1 mM) caused a rapid increase of Pi. Despite recovery of Pi to values exceeding control levels, glycogenolysis only recovered partially, attesting to the Pi-dependence of glycogenolysis, but also to inhibition by aHMol phosphorylation products. However, KCN resulted in conversion of the major part of aHMol1P into aHMol(1,6)P2. Residual inhibition of glycogenolysis was due to aHMol1P. Indeed, the subsequent withdrawal of aHMol caused a further gradual decrease in the proportion of aHMol1P (being converted into aHMol(1,6)P2, in the absence of de novo aHMol1P synthesis), and this resulted in a gradual de-inhibition of glycogenolysis, in the absence of marked changes in Pi. Glycogenolytic rates were consistently predicted by a model assuming non-saturated Pi kinetics and competition by aHMol1P exclusively: In conclusion, limited Pi availability and the presence of competitive inhibitors are decisive factors in the control of the in situ catalytic potential of phosphorylase a.

Phosphonates as 31P-NMR markers of extra- and intracellular space and pH in perfused rat liver.

We evaluated phosphonates (Po) as markers of the extra- and intracellular space in perfused rat liver. (i) In- and outwash behaviour of phenylphosphonate (PhePo), 3-amino-propylphosphonate (NProPo) and methyl phosphonate (MePo) was compared with that of creatine phosphate (CrP), a marker of the extracellular space, and of dimethyl methylphosphonate (MePoMe2), a marker of the total water-accessible space. In- and outwash of CrP was accurately predicted by the time constant (approximately 12 s) for the in- and outwash of inulin, a standard marker of the extracellular space. MePoMe2 rapidly distributed over the total liver volume (about three times the CrP accessible space). PhePo, NProPo and MePo washed rapidly into the extracellular space with CrP, and then steadily spilled over into the MePoMe2-accessible space. Upon outwash, Po signals rapidly declined in phase with that of CrP. Residual Po (PhePo >> NProPo approximately equal to MePo) reflected the amount internalized during prolonged (60 min) inwash. Proportional amounts of residual Po were found in extracts of livers harvested after outwash of perfusate and extracellular markers. Consistent with exclusion from the cells, CrP went undetected in these extracts. (ii) The resonance frequency of residual PhePo after outwash of the extracellular fraction corresponded with the pH reported by cytosolic P1 and responded to transient changes of the intracellular pH, induced by perfusion with and withdrawal of 20 mM NH4Cl. (iii) MePoMe2 homogeneously distributed over perfusate, parenchyma and bile, consistent with unrestricted permeability. Other Po were transported transcellularly and excreted in bile. CrP was virtually excluded from the bile, attesting to a minimal role for 'bulk-phase pinocytotic' transcellular transport, or for 'paracellular' leakage. In summary, charged Po can be used as extracellular markers in liver, provided experimental conditions are adjusted to minimize their internalization. Some Po (e.g. PhePo) can reach intracellular concentrations which suffice for the compound to act as a reporter molecule of the cytosolic pH.

Caffeine counteracts the ergogenic action of muscle creatine loading.

This study aimed to compare the effects of oral creatine (Cr) supplementation with creatine supplementation in combination with caffeine (Cr+C) on muscle phosphocreatine (PCr) level and performance in healthy male volunteers (n = 9). Before and after 6 days of placebo, Cr (0.5 g x kg-1 x day-1), or Cr (0.5 g x kg-1 x day-1) + C (5 mg x kg-1 x day-1) supplementation, 31P-nuclear magnetic resonance spectroscopy of the gastrocnemius muscle and a maximal intermittent exercise fatigue test of the knee extensors on an isokinetic dynamometer were performed. The exercise consisted of three consecutive maximal isometric contractions and three interval series of 90, 80, and 50 maximal voluntary contractions performed with a rest interval of 2 min between the series. Muscle ATP concentration remained constant over the three experimental conditions. Cr and Cr+C increased (P < 0.05) muscle PCr concentration by 4-6%. Dynamic torque production, however, was increased by 10-23% (P < 0.05) by Cr but was not changed by Cr+C. Torque improvement during Cr was most prominent immediately after the 2-min rest between the exercise bouts. The data show that Cr supplementation elevates muscle PCr concentration and markedly improves performance during intense intermittent exercise. This ergogenic effect, however, is completely eliminated by caffeine intake.

Blue form of bacteriorhodopsin and its order-disorder transition during dehydration.

Freshly-prepared blue membranes from Halobacterium halobium, previously reported to be disordered, are shown to have a distinct crystal lattice structure, slightly different from the native form. The lattice of the blue form is disrupted irreversibly when dehydrated. The disorder process was observed using time-resolved small-angle X-ray diffraction and analyzed by radial autocorrelation functions. The diffraction peaks of the in-plane lattice first sharpen and increase due to improved membrane orientation, then the trimer lattice becomes disordered and the unit cell dimension decreases by 1.8 A. In contrast, dehydration of purple membranes does not disorder the lattice, and the unit cell dimension shrinks by only 1.0 A. Comparisons of radial autocorrelation functions for the blue membrane during drying show drastic loss of inter-trimer, long-range correlation while the intra-trimer, short-range correlations remain more or less unchanged. This suggests that the deionized protein trimers can maintain their overall structure during the dehydration, even though the lattice dimension decreases appreciably and the two-dimensional crystallinity is disrupted.

Small-angle x-ray scattering studies of the iron-molybdenum cofactor from Azotobacter vinelandii nitrogenase.

The nitrogenase enzyme complex, consisting of the molybdenum-iron protein and the iron protein, plays a critical role in the biological reduction of dinitrogen to ammonia (nitrogen fixation). The nitrogen-fixing site within the molybdenum-iron protein is an iron-molybdenum-sulfur cofactor (FeMoco) of roughly 1000-2000 Dalton mass. Structural aspects of FeMoco have been determined by spectroscopic and more recently by crystallographic studies. In order to determine the radius of gyration (Rg) of isolated FeMoco, we have performed small-angle x-ray scattering studies of FeMoco in N-methylformamide solution, in the absence of the molybdenum-iron protein. Model compounds of known structure have also been examined in similar solvents, N,N-dimethylformamide and acetonitrile, as controls and for calibration purposes. The Rg values obtained for the models are in good agreement with calculations based upon their respective crystal structures. However, the Rg obtained for FeMoco clearly indicates that the cofactor is not monomeric in solution, but rather aggregated and possibly polydisperse. Further, Rg values were also measured after addition of thiol, dithionite, and thiol and dithionite, to the FeMoco samples. The results indicate, surprisingly, that oxidation state and putative thiol coordination have no detectable effect on the aggregation behavior of FeMoco in solution, as determined by these measurements.