Quantitative data analysis methods for bead-based DNA hybridization assays using generic flow cytometry platforms.

Bead-based assays are in demand for rapid genomic and proteomic assays for both research and clinical purposes. Standard quantitative procedures addressing raw data quality and analysis are required to ensure the data are consistent and reproducible across laboratories independent of flow platform. Quantitative procedures have been introduced spanning raw histogram analysis through to absolute target quantitation. These included models developed to estimate the absolute number of sample molecules bound per bead (Langmuir isotherm), relative quantitative comparisons (two-sided t-tests), and statistical analyses investigating the quality of raw fluorescence data. The absolute target quantitation method revealed a concentration range (below probe saturation) of Cy5-labeled synthetic cytokeratin 19 (K19) RNA of c.a. 1 x 10(4) to 500 x 10(4) molecules/bead, with a binding constant of c.a. 1.6 nM. Raw hybridization frequency histograms were observed to be highly reproducible across 10 triplex assay replicates and only three assay replicates were required to distinguish overlapping peaks representing small sequence mismatches. This study provides a quantitative scheme for determining the absolute target concentration in nucleic acid hybridization reactions and the equilibrium binding constants for individual probe/target pairs. It is envisaged that such studies will form the basis of standard analytical procedures for bead-based cytometry assays to ensure reproducibility in inter- and intra-platform comparisons of data between laboratories.

Selection of a mtDNA sequence variant in hepatocytes of heteroplasmic mice is not due to differences in respiratory chain function or efficiency of replication.

We have previously constructed lines of heteroplasmic mice from two inbred strains (NZB/BinJ and BALB/c) to investigate the mechanisms of segregation of mtDNA sequence variants. Analysis of the segregation behaviour of mtDNA in several tissues showed that the NZB genotype was invariably selected in liver/kidney and the BALB genotype in blood/spleen. Segregation was not significant in post-mitotic tissues. Here we have investigated this novel pattern of mtDNA segregation in isolated hepatocytes to determine the mechanism of selection. Polarographic measurements of respiratory chain function showed no difference between mitochondria containing either 0 or 91-97% NZB mtDNAs on a BALB nuclear background. Single-cell PCR analysis of mtDNA in isolated hepatocytes demonstrated that most hepatocytes eventually fix the NZB genotype. The rate of selection was constant with time and independent of the initial genotype frequency. Based on a mtDNA replication rate of 9.4 days, NZB mtDNA has an approximately 14% selective advantage over BALB mtDNA; however, in vivo pulse labelling with BrdU demonstrated that this was not based on efficiency of replication. Surprisingly, when hepatocytes were cultured in vitro, the majority of independent colonies selected BALB mtDNA, even if they were nearly fixed for the NZB mtDNA genotype when initially plated. These data suggest that selection for NZB mtDNA in the liver of these mice is not based on respiratory chain function at the cellular or organellar level, or a simple replicative advantage, but on a factor(s) involved with mtDNA maintenance.

Correlations of plasma lipid metabolites with hibernation and lactation in wild black bears Ursus americanus.

During the denning period, black bears (Ursus americanus) are capable of enduring several months without food. At the same time, female bears that are pregnant or lactating have an added metabolic stress. Based on laboratory studies, much of the energy required to support metabolism and lactation during denning in black bears comes from lipid reserves. These lipid reserves are mobilized and the most metabolically active lipid fraction in the blood are nonesterified fatty acids (NEFA). Therefore, we hypothesized that plasma NEFAs would be higher in denning relative to active bears and in lactating relative to non-lactating female bears. We further hypothesized that in bears with elevated plasma NEFA levels, other lipid-related parameters (e.g., ketone bodies, albumin, cholesterol, lipase) would also be elevated in the plasma. Denning bears had significantly increased NEFA levels in all classes (saturates, monoenes, and polyenes). A doubling of plasma NEFA levels and a 33% increase in albumin, the plasma fatty acid binding protein, in denning bears, resulted in NEFA/albumin ratios that were higher in denning bears (4:1) compared to those of active bears (3:1). Bears became relatively ketonemic with a 17-fold increase in D-beta-hydroxybutyrate levels during the denning period. Plasma cholesterol approximately doubled and lipase was ten-fold lower in denning relative to active bears. These findings indicate a strong correlation between plasma lipid metabolites and the denning period in a wild population of black bears.

Lactation during hibernation in wild black bears: effects on plasma amino acids and nitrogen metabolites.

This study examined the seasonal and reproductive influences on individual plasma amino acid concentrations and nitrogen metabolites in a black bear population (Ontario, Canada). During hibernation, 11 of 23 plasma amino acids were significantly higher (13%-108%) in lactating than in nonlactating females, without an alteration in plasma total protein or total essential or nonessential amino acid levels. The greatest changes were observed in glutamine, arginine, and glycine levels. Plasma urea, urea/creatinine, and ammonia levels were significantly lower in hibernating compared with active female bears, but lactation had no effect on these parameters. Taken together these results show that lactation during hibernation is an additional metabolic challenge that results in increased mobilization of individual plasma amino acids and no accumulation of nitrogen end products, underlining the remarkable efficiency of amino acid and urea recycling in denning female black bears.

Lipid-DNA complex formation: reorganization and rupture of lipid vesicles in the presence of DNA as observed by cryoelectron microscopy.

Cryoelectron microscopy has been used to study the reorganization of unilamellar cationic lipid vesicles upon the addition of DNA. Unilamellar DNA-coated vesicles, as well as multilamellar DNA lipid complexes, could be observed. Also, DNA induced fusion of unilamellar vesicles was found. DNA appears to adsorb to the oppositely charged lipid bilayer in a monolayer of parallel helices and can act as a molecular "glue" enforcing close apposition of neighboring vesicle membranes. In samples with relatively high DNA content, there is evidence for DNA-induced aggregation and flattening of unilamellar vesicles. In these samples, multilamellar complexes are rare and contain only a small number of lamellae. At lower DNA contents, large multilamellar CL-DNA complexes, often with >10 bilayers, are formed. The multilamellar complexes in both types of sample frequently exhibit partially open bilayer segments on their outside surfaces. DNA seems to accumulate or coil near the edges of such unusually terminated membranes. Multilamellar lipid-DNA complexes appear to form by a mechanism that involves the rupture of an approaching vesicle and subsequent adsorption of its membrane to a "template" vesicle or a lipid-DNA complex.

Influence of acclimation temperature on mitochondrial DNA, RNA, and enzymes in skeletal muscle.

Skeletal muscle fibers typically undergo modifications in their mitochondrial content, concomitant with alterations in oxidative metabolism that occur during the development of muscle fiber and in response to physiological stimuli. We examined how cold acclimation affects the mitochondrial properties of two fish skeletal muscle fiber types and how the regulators of mitochondrial content differed between tissues. After 2 mo of acclimation to either 4 or 18 degrees C, mitochondrial enzyme activities in both red and white muscle were higher in cold-acclimated fish. No significant differences were detected between acclimation temperatures in the abundance of steady-state mitochondrial mRNA (cytochrome-c oxidase 1, subunit 6 of F0F1-ATPase), rRNA (16S), or DNA copy number. Steady-state mRNA for nuclear-encoded respiratory (adenine nucleotide translocase 1) and glycolytic genes showed high interindividual variability, particularly in the cold-acclimated fish. Although mitochondrial enzymes were 10-fold different between the two muscle types, mitochondrial DNA copy number differed only 4-fold. The relative abundance of mitochondrial mRNA and nuclear mRNA in red and white muscle reflected the differences in copy number of their respective genes. These data suggest that the response to physiological stimuli and determination of tissue-specific mitochondrial properties likely result from the regulation of nuclear-encoded genes.

Interactions between bioenergetics and mitochondrial biogenesis.

We studied the interaction between energy metabolism and mitochondrial biogenesis during myogenesis in C2C12 myoblasts. Metabolic rate was nearly constant throughout differentiation, although there was a shift in the relative importance of glycolytic and oxidative metabolism, accompanied by increases in pyruvate dehydrogenase activation state and total activity. These changes in mitochondrial bioenergetic parameters observed during differentiation occurred in the absence of a hypermetabolic stress. A chronic (3 day) energetic stress was imposed on differentiated myotubes using sodium azide to inhibit oxidative metabolism. When used at low concentrations, azide inhibited more than 70% of cytochrome oxidase (COX) activity without changes in bioenergetics (either lactate production or creatine phosphorylation) or mRNA for mitochondrial enzymes. Higher azide concentrations resulted in changes in bioenergetic parameters and increases in steady state COX II mRNA levels. Azide did not affect mtDNA copy number or mRNA levels for other mitochondrial transcripts, suggesting azide affects stability, rather than synthesis, of COX II mRNA. These results indicate that changes in bioenergetics can alter mitochondrial genetic regulation, but that mitochondrial biogenesis accompanying differentiation occurs in the absence of hypermetabolic challenge.

Evidence for three-dimensional interlayer correlations in cationic lipid-DNA complexes as observed by cryo-electron microscopy.

A fingerprint-like pattern across multilamellar, lipid-DNA complexes is attributed to DNA condensed as parallel helices between lipid bilayers. It is argued that the patterning indicates the existence of 3-D correlation forces between DNA-covered bilayers, following the DNA-driven formation of multilamellar liposomes from unilamellar vesicles.

Regulation of muscle mitochondrial design.

Mitochondria are responsible for the generation of ATP to fuel muscle contraction. Hypermetabolic stresses imposed upon muscles can lead to mitochondrial proliferation, but the resulting mitochondria greatly resemble their progenitors. During the mitochondrial biogenesis that accompanies phenotypic adaptation, the stoichiometric relationships between functional elements are preserved through shared sensitivities of respiratory genes to specific transcription factors. Although the properties of muscle mitochondria are generally thought to be highly conserved across species, there are many examples of mitochondrial differences between muscle types, species and developmental states and even within single cells. In this review, we discuss (1) the nature and regulation of gene families that allow coordinated expression of genes for mitochondrial products and (2) the regulatory mechanisms by which mitochondrial differences can arise over physiological and evolutionary time.

Short-term effects of 3,5,3'-triiodothyronine on the intermediary metabolism of the dogfish shark Squalus acanthias: evidence from enzyme activities.

Plasma 3,5,3'-triiodothyronine (T3) concentration decreased significantly (P < 0.05), during 1-5 days of captivity, from levels in the freshly caught dogfish shark Squalus acanthias. The short-term effects of T3 treatment on the intermediary metabolism of S. acanthias were measured in the gill, kidney, liver, and white muscle. Animals were kept for 1-5 days before experimentation. Three hours after an intraperitoneal injection with either a low T3 dose (8.3 pmol T3/kg fish) or a high T3 dose (830 pmol T3/kg fish), selected enzymes of amino acid metabolism, lipid catabolism, ketone body metabolism, glycolysis, and oxidative metabolism were measured. Activity of enzymes of amino acid metabolism and lipid catabolism increased significantly (P < 0.05) in the liver of fish treated with a low T3 dose. The low dose of T3 apparently influences glycolysis as pyruvate kinase activity significantly increase (P < 0.05) in the kidney and white muscle.

Spreading properties of dimyristoyl phosphatidylcholine at the air/water interface.

The spreading behavior of bulk lipid crystals and lipid dispersed in water has been investigated for dimyristoyl phosphatidylcholine at the air/water interface. The stable surface pressures reached with dispersed lipid were found to increase with lipid concentration up to a concentration of approximately 1.2 mg ml-1 where the spreading pressure approached 45 mN m-1, the value for excess lipid crystals placed on the surface (at 30.5 degrees C). These low surface pressures obtained with dispersions are attributed to the existence of 'pre-equilibria': surface pressures that appear steady because of the extremely slow approach to final equilibrium. Attainment of this pre-equilibrium condition usually takes about 20 h, whereas bulk crystals held at the surface generated a high and steady surface pressure within about 1 h. Hydration of the bulk lipid slows down the spreading rate, but does not affect the final surface pressure.