PFA: Program for the Quantitative Assessment of Cell Metabolism by Spectral Data Analysis

Assessment of mitochondrial oxidative metabolism has wide-ranging importance, from pharmacokinetic analysis to studies in cell viability and apoptosis. Here we present the Perfusion File Analyzer (PFA) application for the real-time analysis of spectral data to measure cytochrome c reduction, cytochrome a3 reduction, and other parameters important to cellular metabolism, which are collected during tissue perfusion experiments. Our current efforts are focused on quantitating changes in mitochondrial function by normalizing baseline drift of spectral data while addressing two major challenges: (1) a lack of real-time feedback from the system when aiming is compromised, and (2) an inability to adjust calculated data in the event of spectral shift. PFA has been developed to address these issues, and is currently used for quality assessment of human islets prior to clinical transplantation.     

Gene silencing by S-adenosylmethionine in muscle differentiation.

A well-characterised experimental system, the myogenin gene in C2C12 muscle cell culture, was chosen to better understand the methylation mechanism underlying the regulation of gene expression. We already demonstrated that demethylation dynamics of a specific CpG site in the 5'-flanking region of myogenin well correlates with gene expression and terminal differentiation. Here we demonstrate that S-adenosylmethionine-sulphate-p-toluenesulphonate (SAM) inhibits myogenin expression and myoblast differentiation by delaying the demethylation of specific CpG in differentiating myoblasts. These results suggest new perspectives in methylation mechanisms and the use of SAM in the partial silencing of gene expression, as it could be required in disease treatment.     

Phenotypic and Genetic Analysis of Clock, a New Circadian Rhythm Mutant in Drosophila Melanogaster

Clock is a semidominant X-linked mutation that results in shortening the period of Drosophila melanogaster's free-running locomotor activity rhythm from ca. 24.0 to ca. 22.5 hr. This mutation similarly shortened the phase response curve, determined by resetting activity rhythms with light pulses. Eclosion peaks for Clk cultures were separated by only 22.5 hr instead of the normal 24 hr. Clk was mapped close to, but separable from, another rhythm mutation--period01--by recombination. The estimated distance between these two mutations was short enough to suggest that Clk could be a per allele. If this is the case, the new mutant is unique in that it, unlike other per variants, is associated with essentially normal 1-min courtship song rhythms when Clk is expressed in males. Also, the new rhythm variant could not, in contrast to a short-period per mutation, have its effects on free-running activity rhythms uncovered by deletions. This result, and the lack of coverage of Clk's effects by duplications, suggest that it is not a simple hypomorphic or amorphic mutation.     

Nucleotide analogue inhibitors of purine nucleoside phosphorylase

The diphosphate of the antiherpetic agent acyclovir [9-[(2-hydroxyethoxy)methyl]guanine] has been shown to inhibit purine nucleoside phosphorylase with unique potency (Tuttle, J. V., and Krenitsky, T. A. (1984) J. Biol. Chem. 259, 4065-4069). A major factor contributing to the superior inhibition by this diphosphate over the corresponding mono- and triphosphates is revealed here. Homologues of acyclovir mono- and diphosphate that extend the ethoxy moiety by one to four methylene groups were synthesized. These homologues were evaluated for their ability to inhibit human purine nucleoside phosphorylase. Within the diphosphate series, the Ki values increased progressively with increasing chain length. With the monophosphates, the Ki values reached a minimum with the homologue containing a pentoxy moiety. A plot of chain length versus Ki values for both mono- and diphosphates showed that both series had similar optimal distances between the aminal carbon and the terminal oxygen anion. Monophosphates with optimal positioning were somewhat less potent than diphosphates with similar positioning. Nevertheless, it was clear that a major factor in determining potency of inhibition was the distance of the terminal phosphate from the guanine moiety.