Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae

A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, approximately 10{sup 8} yeast cells were harvested by centrifugation and then lysed under non-oxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50-mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH{sub 3}CN + 50-mM ammonium acetate (3:1; v:v) was added to the cell lysates. After sample centrifugation to pellet precipitated proteins, organic solvent removal was performed on supernatants by chloroform extraction. The remaining aqueous phase was dried and resuspended in 50-mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-VIS absorbance detection. Applicability of this procedure for quantifying NAD and NADH levels was evaluated by culturing yeast under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. NAD and NADH contents are similar to previously reported levels in yeast obtained using enzymatic assays performed separately on acid (for NAD) and alkali (for NADH) extracts. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (1) applicable to quantification of these metabolites in mammalian and bacterial cell cultures; and (2) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures

Immunohistochemical detection and regulation of α5 nicotinic acetylcholine receptor (nAChR) subunits by FoxA2 during mouse lung organogenesis

<p>Abstract</p> <p>Background</p> <p>α₅nicotinic acetylcholine receptor (nAChR) subunits structurally stabilize functional nAChRs in many non-neuronal tissue types. The expression of α₅nAChR subunits and cell-specific markers were assessed during lung morphogenesis by co-localizing immunohistochemistry from embryonic day (E) 13.5 to post natal day (PN) 20. Transcriptional control of α₅nAChR expression by FoxA2 and GATA-6 was determined by reporter gene assays.</p> <p>Results</p> <p>Steady expression of α₅nAChR subunits was observed in distal lung epithelial cells during development while proximal lung expression significantly alternates between abundant prenatal expression, absence at PN4 and PN10, and a return to intense expression at PN20. α₅expression was most abundant on luminal edges of alveolar type (AT) I and ATII cells, non-ciliated Clara cells, and ciliated cells in the proximal lung at various periods of lung formation. Expression of α₅nAChR subunits correlated with cell differentiation and reporter gene assays suggest expression of α₅is regulated in part by FoxA2, with possible cooperation by GATA-6.</p> <p>Conclusions</p> <p>Our data reveal a highly regulated temporal-spatial pattern of α₅nAChR subunit expression during important periods of lung morphogenesis. Due to specific regulation by FoxA2 and distinct identification of α₅in alveolar epithelium and Clara cells, future studies may identify possible mechanisms of cell differentiation and lung homeostasis mediated at least in part by α₅-containing nAChRs.</p

At the Biological Modeling and Simulation Frontier

We provide a rationale for and describe examples of synthetic modeling and simulation (M&S) of biological systems. We explain how synthetic methods are distinct from familiar inductive methods. Synthetic M&S is a means to better understand the mechanisms that generate normal and disease-related phenomena observed in research, and how compounds of interest interact with them to alter phenomena. An objective is to build better, working hypotheses of plausible mechanisms. A synthetic model is an extant hypothesis: execution produces an observable mechanism and phenomena. Mobile objects representing compounds carry information enabling components to distinguish between them and react accordingly when different compounds are studied simultaneously. We argue that the familiar inductive approaches contribute to the general inefficiencies being experienced by pharmaceutical R&D, and that use of synthetic approaches accelerates and improves R&D decision-making and thus the drug development process. A reason is that synthetic models encourage and facilitate abductive scientific reasoning, a primary means of knowledge creation and creative cognition. When synthetic models are executed, we observe different aspects of knowledge in action from different perspectives. These models can be tuned to reflect differences in experimental conditions and individuals, making translational research more concrete while moving us closer to personalized medicine

Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae Seperation Science Single sample extraction and HPLC processing for quantification of 1 NAD and NADH levels in Saccharomyces cerevisiae 2 3

Abstract 28 A robust redox extraction protocol for quantitative and reproducible metabolite isolation and 29 recovery has been developed for simultaneous measurement of nicotinamide adenine 30 dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following 31 culture in liquid media, approximately 10 8 yeast cells were harvested by centrifugation and then 32 lysed under non-oxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50-mM 33 ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH 3 CN + 50-mM 34 ammonium acetate (3:1; v:v) was added to the cell lysates. After sample centrifugation to pellet 35 precipitated proteins, organic solvent removal was performed on supernatants by chloroform 36 extraction. The remaining aqueous phase was dried and resuspended in 50-mM ammonium 37 acetate. NAD and NADH were separated by HPLC and quantified using UV-VIS absorbance 38 detection. Applicability of this procedure for quantifying NAD and NADH levels was evaluated 39 by culturing yeast under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. 40 NAD and NADH contents are similar to previously reported levels in yeast obtained using 41 enzymatic assays performed separately on acid (for NAD) and alkali (for NADH) extracts. 42 Results demonstrate that it is possible to perform a single preparation to reliably and robustly 43 quantitate both NAD and NADH contents in the same sample. Robustness of the protocol 44 suggests it will be 1) applicable to quantification of these metabolites in mammalian and 45 bacterial cell cultures; and 2) amenable to isotope labeling strategies to determine the relative 46 contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures. 47 48