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In JoVE (1)
Other Publications (8)
Articles by James D. Shoemaker in JoVE
JoVE General
One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure
The urease method of sample preparation for GC/MS analysis of intermediary metabolites is presented by its inventor. The method allows one-step follow-up of newborn screening for inborn errors by tandem mass spectrometry by quantifying carbohydrates, organic and amino acids all in a single process.
Other articles by James D. Shoemaker on PubMed
Disruption of Aldehyde Reductase Increases Group Size in Dictyostelium
Developing Dictyostelium cells form structures containing approximately 20,000 cells. The size regulation mechanism involves a secreted counting factor (CF) repressing cytosolic glucose levels. Glucose or a glucose metabolite affects cell-cell adhesion and motility; these in turn affect whether a group stays together, loses cells, or even breaks up. NADPH-coupled aldehyde reductase reduces a wide variety of aldehydes to the corresponding alcohols, including converting glucose to sorbitol. The levels of this enzyme previously appeared to be regulated by CF. We find that disrupting alrA, the gene encoding aldehyde reductase, results in the loss of alrA mRNA and AlrA protein and a decrease in the ability of cell lysates to reduce both glyceraldehyde and glucose in an NADPH-coupled reaction. Counterintuitively, alrA- cells grow normally and have decreased glucose levels compared with parental cells. The alrA- cells form long unbroken streams and huge groups. Expression of AlrA in alrA- cells causes cells to form normal fruiting bodies, indicating that AlrA affects group size. alrA- cells have normal adhesion but a reduced motility, and computer simulations suggest that this could indeed result in the formation of large groups. alrA- cells secrete low levels of countin and CF50, two components of CF, and this could partially account for why alrA- cells form large groups. alrA- cells are responsive to CF and are partially responsive to recombinant countin and CF50, suggesting that disrupting alrA inhibits but does not completely block the CF signal transduction pathway. Gas chromatography/mass spectroscopy indicates that the concentrations of several metabolites are altered in alrA- cells, suggesting that the Dictyostelium aldehyde reductase affects several metabolic pathways in addition to converting glucose to sorbitol. Together, our data suggest that disrupting alrA affects CF secretion, causes many effects on cellular metabolism, and has a major effect on group size.
A Folate-dependent Metabolite in Amniotic Fluid from Pregnancies with Normal or Trisomy 21 Chromosomes
Previous studies have given conflicting results as to whether or not folate metabolism is altered in Down syndrome. Folate is necessary to facilitate metabolism of one-carbon units. Folate accepts one-carbon units from one-carbon unit donors, including formiminoglutamate (FIGLU). Folate deficiency leads to accumulation of FIGLU and impairment of one-carbon unit metabolism. FIGLU is a functional measure of folate deficiency.
Feeding Drosophila a Biotin-deficient Diet for Multiple Generations Increases Stress Resistance and Lifespan and Alters Gene Expression and Histone Biotinylation Patterns
Energy restriction increases stress resistance and lifespan in Drosophila melanogaster and other species. The roles of individual nutrients in stress resistance and longevity are largely unknown. The vitamin biotin is a potential candidate for mediating these effects, given its known roles in stress signaling and gene regulation by epigenetic mechanisms, i.e. biotinylation of histones. Here, we tested the hypothesis that prolonged culture of Drosophila on biotin-deficient (BD) medium increases stress resistance and lifespan. Flies were fed a BD diet for multiple generations; controls were fed a biotin-normal diet. In some experiments, a third group of flies was fed a BD diet for 12 generations and then switched to control diets for 2 generations to eliminate potential effects of short-term biotin deficiency. Flies fed a BD diet exhibited a 30% increase in lifespan. This increase was associated with enhanced resistance to the DNA-damaging agent hydroxyurea and heat stress. Also, fertility increased significantly compared with biotin-normal controls. Biotinylation of histones was barely detectable in biotin-deprived flies, suggesting that epigenetic events might have contributed to effects of biotin deprivation.
Organic Acid Concentrations in Amniotic Fluid Found in Normal and Down Syndrome Pregnancies
Organic acids were examined from normal and Down syndrome pregnancies to identify possible differences between the amniotic fluid from fetuses with Down Syndrome compared with that of normal fetuses.
Pyridoxine-related Metabolite Concentrations in Normal and Down Syndrome Amniotic Fluid
Some studies of children with Down syndrome have found mild abnormalities in the metabolism of pyridoxine (vitamin B(6)); therefore the present question is whether such abnormalities might also be present in the amniotic fluid of fetuses with Down syndrome.
Isocitrate Dehydrogenase is Important for Nitrosative Stress Resistance in Cryptococcus Neoformans, but Oxidative Stress Resistance is Not Dependent on Glucose-6-phosphate Dehydrogenase
The opportunistic intracellular fungal pathogen Cryptococcus neoformans depends on many antioxidant and denitrosylating proteins and pathways for virulence in the immunocompromised host. These include the glutathione and thioredoxin pathways, thiol peroxidase, cytochrome c peroxidase, and flavohemoglobin denitrosylase. All of these ultimately depend on NADPH for either catalytic activity or maintenance of a reduced, functional form. The need for NADPH during oxidative stress is well established in many systems, but a role in resistance to nitrosative stress has not been as well characterized. In this study we investigated the roles of two sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1) and NADP(+)-dependent isocitrate dehydrogenase (Idp1), in production of NADPH and resistance to oxidative and nitrosative stress. Deletion of ZWF1 in C. neoformans did not result in an oxidative stress sensitivity phenotype or changes in the amount of NADPH produced during oxidative stress compared to those for the wild type. Deletion of IDP1 resulted in greater sensitivity to nitrosative stress than to oxidative stress. The amount of NADPH increased 2-fold over that in the wild type during nitrosative stress, and yet the idp1Delta strain accumulated more mitochondrial damage than the wild type during nitrosative stress. This is the first report of the importance of Idp1 and NADPH for nitrosative stress resistance.
Substrate Oxidation and Cardiac Performance During Exercise in Disorders of Long Chain Fatty Acid Oxidation
The use of long-chain fatty acids (LCFAs) for energy is inhibited in inherited disorders of long-chain fatty acid oxidation (FAO). Increased energy demands during exercise can lead to cardiomyopathy and rhabdomyolysis. Medium-chain triglycerides (MCTs) bypass the block in long-chain FAO and may provide an alternative energy substrate to exercising muscle.
