No abstract

DNA replication occurs almost exclusively during S-phase of the cell cycle and represents a simple biochemical metric of cell division. Previous methods for measuring cell proliferation rates have important limitations. Here, we describe experimental protocols for measuring cell proliferation and death rates based on the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells. Label incorporation is measured by gas chromatography/mass spectrometry. Modifications of the basic protocol permit analysis of small cell samples (down to 2,000 cells). The theoretical basis and operational requirements for effective use of these methods to measure proliferation and death rates of cells in vivo are described. These methods are safe for use in humans, have technical and interpretation advantages over alternative techniques and can be used on small numbers of cells. The protocols enable definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans.

Mutations in copper/zinc superoxide dismutase 1 (SOD1), a genetic cause of human amyotrophic lateral sclerosis, trigger motoneuron death through unknown toxic mechanisms. We report that transgenic SOD1G93A mice exhibit striking and progressive changes in neuronal microtubule dynamics from an early age, associated with impaired axonal transport. Pharmacologic administration of a microtubule-modulating agent alone or in combination with a neuroprotective drug to symptomatic SOD1G93A mice reduced microtubule turnover, preserved spinal cord neurons, normalized axonal transport kinetics, and delayed the onset of symptoms, while prolonging life by up to 26%. The degree of reduction of microtubule turnover was highly predictive of clinical responses to different treatments. These data are consistent with the hypothesis that hyperdynamic microtubules impair axonal transport and accelerate motor neuron degeneration in amyotrophic lateral sclerosis. Measurement of microtubule dynamics in vivo provides a sensitive biomarker of disease activity and therapeutic response and represents a new pharmacologic target in neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS)2 is a late-onset, progressive neurodegenerative disease affecting motoneurons (1). The etiology of the majority of ALS cases is unknown, but ϳ20% of familial disease cases are due to mutations in copper-zinc superoxide dismutase1 (SOD1) (2). This led to the development of SOD1 transgenic mice as models of disease (2-5).Physically, motoneurons are unique, representing the longest cells in the body, with axons of some motoneurons in the spinal cord extending a meter or more to reach an end organ. As a result of this morphology, exceptional demands are placed on motoneurons. Active transport along lengthy axons is required to convey newly made materials from the cell body to the farthest nerve endings, and to convey nutrients and metabolites back to the cell body. Microtubules are an essential component of the neuron's scaffold and represent the "roadway," or conveyer belt, that neurons use to transport nutrients (6 -10). Microtubule-based transport is mandatory for survival of motoneurons and muscle cells; changes in slow axonal transport have been linked to neuropathogenesis in mutant SOD1 transgenic mice (11-13). In addition, the assembly and disassembly of microtubule polymers in motoneurons is highly responsive to cellular insults, such as excitotoxic stimuli (8, 14 -16).The relation between dynamics of microtubules and neuronal pathogenesis has not been explored in detail, in part due to limited techniques for measuring microtubule dynamics in vivo. In most non-neuronal cells, tubulin dimers and microtubule polymers exist in rapid dynamic equilibrium, as we have recently shown in vivo by isotopic labeling (17). In neurons, however, this rapid turnover of axonal and dendritic microtubules is believed to be less dynamic due to their interactions with a specific subclass of microtubule-associated proteins (MAPs) (18 -20). This stability of microtubule...

A preliminary investigation of the electrochemical deposition of Pt, Pb, and Hg adlayers on conductive diamond thin-film surfaces has been made using cyclic voltammetry and scanning electron microscopy. The diamond thin films employed were polycrystalline, grown on conductive Si substrates (1 cm 2) to a thickness of ca. 14 i~m, and doped with boron at a nominal atomic concentration ranging between 10 ~9 and 102~ cm -3. The cyclic volammetric measurements were performed both in a conventional glass electrochemical cell and in a thin-layer flow cell. The results demonstrate that metaUization of diamond film surfaces electrochemically is feasible, opening the door for the development of novel catalytic electrodes, sensors, and detectors using this advanced material.

Background--Reverse cholesterol transport from peripheral tissues is considered the principal atheroprotective mechanism of high-density lipoprotein, but quantifying reverse cholesterol transport in humans in vivo remains a challenge. We describe here a method for measuring flux of cholesterol though 3 primary components of the reverse cholesterol transport pathway in vivo in humans: tissue free cholesterol (FC) efflux, esterification of FC in plasma, and fecal sterol excretion of plasma-derived FC.Methods and Results--A constant infusion of [2,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]-cholesterol was administered to healthy volunteers. Three-compartment SAAM II (Simulation, Analysis, and Modeling software; SAAM Institute, University of Washington, WA) fits were applied to plasma FC, red blood cell FC, and plasma cholesterol ester 13 C-enrichment profiles. Fecal sterol excretion of plasma-derived FC was quantified from fractional recovery of intravenous [2,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]-cholesterol in feces over 7 days. We examined the key assumptions of the method and evaluated the optimal clinical protocol and approach to data analysis and modeling. A total of 17 subjects from 2 study sites (n=12 from first site, age 21 to 75 years, 2 women; n=5 from second site, age 18 to 70 years, 2 women) were studied. Tissue FC efflux was 3.79±0.88 mg/kg per hour (mean ± standard deviation), or %8 g/d. Red blood cell-derived flux into plasma FC was 3.38±1.10 mg/kg per hour. Esterification of plasma FC was %28% of tissue FC efflux (1.10±0.38 mg/kg per hour). Recoveries were 7% and 12% of administered [2,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]-cholesterol in fecal bile acids and neutral sterols, respectively.Conclusions--Three components of systemic reverse cholesterol transport can be quantified, allowing dissection of this important function of high-density lipoprotein in vivo. Effects of lipoproteins, genetic mutations, lifestyle changes, and drugs on these components can be assessed in humans. ( J Am Heart Assoc. 2012;1:e001826 doi: 10.1161/JAHA.112.001826)Key Words: cholesterol efflux • esterification • reverse cholesterol transport • isotope labeling, stable • sterol excretion T he regulation of cellular cholesterol homeostasis is crucial for membrane function and cell survival and is maintained by multiple mechanisms, including control of uptake, synthesis, storage, and efflux. Compared to the pathways of cellular uptake and de novo synthesis of cholesterol, however, less information exists about the control of flux though pathways that remove cholesterol from cells and from the whole organism, 1,2 particularly in humans. [3][4][5] These pathways collectively have been termed reverse cholesterol transport (RCT). RCT is postulated to play a fundamental role in cholesterol homeostasis and distribution among tissues 5 and thereby in the development and reversal of atherosclerosis. 6,7 The atheroprotective effects of high-density lipoprotein cholesterol (HDL-C) in both human and animal studies often hav...