Rhabdomyolysis is a severe adverse effect of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins). This myopathy is strongly enhanced by the combination with statins and fibrates, another hypolipidaemic agent. We have evaluated the initial step of statin-induced apoptosis by the detection of membrane flip-flop using flow cytometric analysis. L6 rat myoblasts were treated with various statins (atorvastatin (3 microM), cerivastatin (3 microM), fluvastatin (3 microM), pravastatin (3 mM), or simvastatin (3 microM)) for 2, 4 or 6 h followed by reacting with FITC-conjugated annexin V for the detection of initial apoptosis signal (flip-flop). Various statin-treated myoblasts were significantly stained with FITC-annexin V at 6 h, whereas they were not detected at 2 h. Moreover, immunoblot analysis indicated that when the cells were treated with cerivastatin (3 microM), membrane-associated Ras protein was activated and detached until 6 h, resulting in cell death through the consequent activation of caspase-8. On the other hand, since cytosolic Ras activation did not activate, there is still an unknown mechanism in statin-related Ras depletion. In conclusion, statin-induced apoptosis in muscular tissue was directly initiated by the farnesyl-anchored Ras protein depletion from cell membrane with subsequent apoptosis.

Rhabdomyolysis is a severe adverse effect of hypolipidaemic agents such as statins and fibrates. We evaluated this muscular cytotoxicity with an in-vitro culture system. Cellular apoptosis was determined using phase-contrast and fluorescein microscopic observation with Hoechst 33342 staining. L6 rat myoblasts were treated with various statins and bezafibrate under various conditions. With statins only, skeletal cytotoxicity was ranked as cerivastatin > fluvastatin > simvastatin > atorvastatin > pravastatin in order of decreasing potency. Combined application of fibrates enhanced atorvastatin-induced myopathy, which causes little apoptosis alone. These results suggest that statins and fibrates synergistically aggravate rhabdomyolysis.

The biosynthetic route of the pyrimidine moiety of thiamin is different in prokaryotes and eukaryotes. In prokaryotes, the pyrimidine moiety is synthesized from aminoimidazole ribonucleotide, an intermediate of purine biosynthesis, while in eukaryotes, we have reported that the N-1, C-2, and N-3 atoms of the imidazole ring of histidine are incorporated into N-3, C-4, and the amino group attached to the C-4 atoms of the pyrimidine moiety, respectively, as a unit; the rest of the atoms of the pyrimidine moiety originate from pyridoxine as a unit. It has been reported that urocanic acid, the deaminated compound of histidine, is the direct precursor of the pyrimidine moiety. In the present report, we have investigated whether histidine or urocanic acid is the direct precursor of the pyrimidine moiety in Saccharomyces cerevisiae , using tracer experiments with 1) 13 C-formate and urocanic acid, 2) 15 N-NH 4 Cl and urocanic acid, 3) 15 N-NH 4 Cl and histidine, and 4) 13 C-histidine and urocanic acid. The GC-MS analysis revealed that the incorporation of the 15 N atom of 15 NH 4 Cl was not affected by the presence of urocanic acid, although it was affected by histidine, and the incorporation of 13 C-histidine was not affected by the presence of urocanic acid. These results confirm that histidine is the direct precursor of the pyrimidine moiety of thiamin in S. cerevisiae .

Summary It is well known that some amino acids inhibit bacterial growth. L -Serine is known to inhibit the growth of Escherichia coli by inhibition of homoserine dehydrogenase (EC 1.1.1.3). It has been reported that this L -serine inhibition may be prevented by the addition of L -isoleucine or L -threonine to the medium. In our study, however, recovery of the growth inhibition of Escherichia coli by L -serine occurred in the presence of several amino acids, especially L -phenylalanine. In an attempt to further elucidate this inhibition mechanism, different intermediates of aromatic amino acid biosynthesis were added to the growth medium. Recovery from the inhibition did not occur in the presence of prephenate but did occur when phenylpyruvate was added to the medium. The specific activity of prephenate dehydratase decreased in cells grown in the presence of L -serine. However, L -serine did not inhibit in vitro prephenate dehydratase activity, and the expression of pheA, which encodes the prephenate dehydratase, was not depressed by L -serine. We suggest that L -serine acts via another inhibition mechanism. Although this inhibition mechanism has not been fully elucidated, our results suggest that the addition of L -serine to the growth medium inhibits prephenate dehydratase synthesis and thus affects L -phenylalanine biosynthesis.

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