Stephen P. F. Miller

Type C Niemann-Pick Disease: Use of Hydrophobic Amines to Study Defective Cholesterol Transport

Niemann-Pick Type C (NPC) disease is a cholesterol lipidosis resulting from defective postlysosomal cholesterol transport. In normal cells this segment of cholesterol trafficking is inhibited by treatment with either U18666A or imipramine. Other compounds are also capable of blocking postlysosomal cholesterol transport: stearylamine, RV-538, and sphinganine inhibit low-density lipoprotein-induced esterification of cholesterol and cause unesterified cholesterol to accumulate in perinuclear vesicles. These vesicles can be stained with filipin to give a staining pattern indistinguishable from that seen in NPC fibroblasts. Because all of these compounds are hydrophobic amines, we conclude that most, if not all, hydrophobic amines block the postlysosomal transport of cholesterol. These results also raise the possibility that an endogenous amine, e.g., sphinganine, may inhibit cholesterol transport in NPC.

Type C Niemann-Pick disease: a murine model of the lysosomal cholesterol lipidosis accumulates sphingosine and sphinganine in liver.

We have determined the levels of free sphingoid bases in livers of normal and cholesterol lipidotic Niemann-Pick type C mice. Hepatic sphingosine and sphinganine levels in affected mice (593 pmol/mg protein) were elevated more than 20-fold when compared to levels in age-matched normal mice (26 pmol/mg protein). Upon fractionation of mutant liver homogenates by differential centrifugation, most of the sphingoid bases sedimented with beta-hexosaminidase in the 9000 x g pellet. Co-sedimentation of sphingoid bases with a lighter beta-hexosaminidase peak in Percoll gradients suggests that these bases accumulate in lipid laden lysosomes. A cytosolic sphinganine kinase is the first enzyme in the degradative pathway of sphingoid base metabolism. Activity of this enzyme was partially deficient in crude mutant liver cytosolic extracts due to the presence of an inhibitory substance. Following molecular sieving of mutant cytosolic extracts on Sepharose 4B, sphinganine kinase, with normal levels of activity, was resolved from a complex higher-molecular-weight inhibitor fraction. The Km values for either sphinganine or ATP-Mg substrates with partially purified sphinganine kinase from normal and mutant mouse liver extracts, were similar. These findings indicate that accumulation of free sphingoid bases is not due to a direct inherent deficiency in the catalytic activity of sphinganine kinase. The possible cause and effect relationship between the accumulation of these endogenous hydrophobic amines and the lesion in intracellular cholesterol trafficking in Niemann-Pick type C disease is discussed.

The chemical identification of the granary weevil aggregation pheromone

Abstract (R*,S*)-1-Ethylpropyl 2-methyl-3-hydroxypentanoate is identified as the major component of the aggregation pheromone of Sitophilus granarius (L.), the granary weevil.

Synthesis of an inhibitor of sphingosine-1-phosphate lyase

Abstract The 2-vinyl dihydrosphingosine-1-phosphate 1 was synthesized and tested for inhibition of sphingosine-1-phosphate lyase. The carbon skeleton was prepared by acylation of a methionine imine with N-methoxy-N-methyl palmitamide. The vinyl group was generated by thermal sulfoxide elimination. Reduction of both carbonyl groups and deprotection gave 2-vinyl dihydrosphingosine. The phosphate group was introduced by means of bis(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite as monofunctional phosphitylation reagent.

Structural elucidation of alfalfa root saponins by mass spectrometry and nuclear magnetic resonance analysis

Structures of saponins may be fully elucidated using a combination of n.m.r. and m.s. instead of the usual degradation techniques. Well resolved proton n.m.r. spectra and chromatographic separation can be obtained from peracetylated mixtures of saponins. Under these conditions, genins are identified mainly by 13C n.m.r. spectroscopy; sugars and their points of attachment are determined by examining 1H chemical shifts and coupling constants following assignments made by 2D COSY and relayed COSY experiments. Sugar chains are sequenced by observing intersugar connectivities through long-range couplings (delayed COSY) or n.o.e. Alternatively, the use of Californium plasma desorption m.s. provides quasimolecular ions and fragments corresponding to the sequential rupture of the chain of sugars. Free carboxylic acids are located on an acetylated saponin by looking at the disappearance, in the presence of Eu complexes, of the carbon situated α to the carbonyl in the 13C n.m.r. spectrum. The above methods have been employed to determine the structures of the seven major saponins of alfalfa root, which are allegedly responsible for the antifeedant properties of the vegetable.

Synthesis and inhibitory activity of difluoroketone substrate analogs of N-myristoyltransferase.

Abstract Two fluorinated nonhydrolyzable analogs of myristoyl-coenzyme A were synthesized and tested for inhibitory activity against N-myristoyltransferase (NMT).S-(2,2-Difluoro-3-oxohexadecyl)-coenzyme A (3) and S-(3,3-difluoro-2-oxopentadecyl)-coenzyme A (2) were prepared by alkylation of coenzyme A and were purified by reverse phase chromatography. Inhibition of NMT was observed with 3 and 2, with IC50s of 110 nM and 80 nM, respectively, in an in vitro assay developed in our laboratory. The known unfluorinated analog S-(2-oxopentadecyl)-coenzyme A (1) was found to have an IC50 of 7 nM. At 100 μM in D2O, 3 was 59% hydrated and 2 was 88% hydrated.