Dominant non-coding repeat expansions in human disease.

The general model that dominant diseases are caused by mutations that result in a gain or change in function of the corresponding protein was challenged by the discovery that the myotonic dystrophy type 1 mutation is a CTG expansion located in the 3' untranslated portion of a kinase gene. The subsequent discovery that a similar transcribed but untranslated CCTG expansion in an intron causes the same multisystemic features in myotonic dystrophy type 2 (DM2), along with other developments in the DM1 field, demonstrate a mechanism in which these expansion mutations cause disease through a gain of function mechanism triggered by the accumulation of transcripts containing CUG or CCUG repeat expansions. A similar RNA gain of function mechanism has also been implicated in fragile X tremor ataxia syndrome (FXTAS) and may play a role in pathogenesis of other non-coding repeat expansion diseases, including spinocerebellar ataxia type 8 (SCA8), SCA10, SCA12 and Huntington disease-like 2.

(R)-, (S)-, and racemic fluoxetine N-demethylation by human cytochrome P450 enzymes.

Fluoxetine is one of the most widely prescribed selective serotonin reuptake inhibitors (SSRIs) that is marketed worldwide. However, details of its human hepatic metabolism have been speculative and incomplete, possibly due to the sensitivity of analytical techniques and selectivity of specific in vitro probes and reagents used. Studies with (R)-, (S)-, and racemic fluoxetine were undertaken to determine the stereospecific nature of its metabolism and estimate intrinsic clearance contributions of each CYP for fluoxetine N-demethylation. Measurable fluoxetine N-demethylase activity was catalyzed by CYP1A2, -2B6, -2C9, -2C19, -2D6, -3A4, and -3A5. All enzymes catalyzed this reaction for both enantiomers and the racemate, and intrinsic clearance values were similar for the enantiomers for all CYP enzymes except CYP2C9, which demonstrated stereoselectivity for R- over the S-enantiomer. Scaling the intrinsic clearance values for the individual CYP enzymes to estimate contributions of each in human liver microsomes suggested that CYP2D6, CYP2C9, and CYP3A4 contribute the greatest amount of fluoxetine N-demethylation in human liver microsomes. These data were corroborated with the examination of the effects of CYP-specific inhibitors quinidine (CYP2D6), sulfaphenazole (CYP2C9), and ketoconazole (CYP3A4) on fluoxetine N-demethylation in pooled human liver microsomes. Together, these findings suggest a significant role for the polymorphically expressed CYP2D6 in fluoxetine clearance and are consistent with reports on the clinical pharmacokinetics of fluoxetine.

The tracing of the pathway of mevalonate's metabolism to other than sterols.

Specifically 14C-labeled mevalonic acids were administered to rats in diabetic ketosis, and the distribution of 14C was determined in the hydroxybutyric acid each rat excreted. Also, the distributions of 14C were determined in hydroxybutyric acid formed by slices of livers and kidneys from rats in diabetic ketosis and incubated with the specifically labeled mevalonic acids. The distributions found are in accord with the conversion of mevalonate to hydroxymethylglutaryl-CoA by the shunt pathway proposed by J. Edmond and G. Popják ((1974) J. Biol. Chem. 249, 66-71). That is, carbon 5 of mevalonate was metabolized to form the carboxyl of acetyl-CoA and carbons 2 and 3 of mevalonate were converted in large measure to hydroxybutyric acid without acetyl-CoA as an intermediate, i.e. the bond between carbon 2 and 3 was not cleaved, while the bond between 1 and 2, traced with [1,2-14C]mevalonate, was cleaved. Similar distributions of 14C were found in hydroxybutyric acid excreted by rats in diabetic ketosis administered specifically 14C-labeled isovaleric acids, isovaleric acid having in its metabolism intermediates common to those in the shunt pathway.

A method for quantitating the contributions of the pathways of acetoacetate formation and its application to diabetic ketosis in vivo.

A method has been developed for estimating in the intact cell the contribution of deacylation of acetoacetyl-CoA to the formation of acetoacetate relative to acetoacetate's formation via hydroxymethylglutaryl (HMG)-CoA. Estimates depend upon the fraction of the terminal four carbons of an even carbon-containing fatty acid that are converted to acetoacetate without prior conversion to acetyl-CoA, since in the formation of acetoacetate via HMG-CoA the omega-2 and omega-3 carbons of the fatty acid are converted to acetyl-CoA. Incorporation of 14C from [16-14C]palmitic acid into carbon 2 relative to carbon 4 of acetoacetate is used as the measure of the formation of the acetoacetate from the omega and omega-1 carbons of the fatty acid without acetyl-CoA as an intermediate. Incorporation of 14C from [13-14C]palmitic acid into carbon 1 relative to carbon 3 of acetoacetate is the measure of the formation of acetoacetate from the omega-2 and omega-3 carbons without acetyl-CoA as an intermediate. Comparison of these incorporations is made with incorporation into the carbons of acetoacetate of 14C from palmitic acid labeled with 14C in any of its first 12 carbons since such incorporation must proceed via acetyl-CoA as an intermediate. In an application of this approach, the specifically 14C-labeled palmitic acids were injected into rats in diabetic ketosis. Hydroxybutyric acid that each rat excreted was isolated and degraded. From the ratios of incorporation into the carbons of the hydroxybutyrates, as a minimum, 11% of the total quantity of hydroxybutyrate excreted by the rats was formed from acetoacetyl-CoA without HMG-CoA as an intermediate.

Pathways of acetoacetate's formation in liver and kidney.

Specifically 14C-labeled palmitic acids were perfused through livers and incubated with slices of kidneys from rats in diabetic ketosis. The distribution of 14C in the hydroxybutyric acid formed was determined. In liver, the ratio of incorporation of 14C from [13-14C]palmitic acid into carbon 1 to carbon 3 of the hydroxybutyric acid was the same as the ratio in carbon 2 to carbon 4 from [6-14C]palmitic acid. In kidney, the carbon 1-to-carbon 3 ratio was more than twice the carbon 2-to-carbon 4 ratio. In both tissues, 14C from [16-14C] palmitic acid was preferentially incorporated into carbon 4 compared to carbon 2 of the hydroxybutyric acid, but more so in liver than kidney. These results mean that in liver, the sole pathway of acetoacetate formation is via hydroxymethylglutaryl-CoA, while in kidney it is not. Rather in kidney, acetoacetyl-CoA is converted to acetoacetate to a large extent by direct deacylation, presumably via a transferase- and/or deacylase-catalyzed reaction. In liver, most of the palmitic acid utilized is converted to acetoacetate while in kidney it is not. We previously estimated that, as a minimum, 11% of the hydroxybutyric acid excreted by the rat in diabetic ketosis is formed without hydroxymethylglutaryl-CoA as an intermediate. The kidney appears to be the source of this hydroxybutyric acid if the pathways operative in these tissues in vitro are those that also operate in vivo.

Cytophilic macroglobulin reactive with bacterial protein in mice immunized with ribonucleic acid-protein fractions of virulent Salmonella typhimurium.

Cytophilic macroglobulin was eluted (by heating at 56 C for 60 min) from lymphoid cells of mice immunized with subfractions of virulent Salmonella typhimurium SR-11 or vaccinated with an attenuated strain of S. typhimurium (RIA). No such macroglobulin was present on comparable cell populations from normal or adjuvant-injected mice. Cytophilic macroglobulin was also present in the sera of immunized mice. In mice immunized with RNA and protein-containing subfractions extracted from lysates of S. typhimurium SR-11, the cytophilic macroglobulin was present by 14 days after immunization, whereas it was present by 10 days in mice injected with living attenuated S. typhimurium RIA. This macroglobulin reacts specifically with protein-rich components present in the immunogenic subfractions of the whole bacterial cells and has been demonstrated to facilitate phagocytic uptake of virulent S. typhimurium SR-11. Moreover, a similar protein material was present in broth culture media in which the salmonellae were cultivated. Greater amounts were detectable in older (4 day) cultures.