A Kruppel-like factor KLF15 contributes fasting-induced transcriptional activation of mitochondrial acetyl-CoA synthetase gene AceCS2.

Acetyl-CoA synthetase 2 (AceCS2) produces acetyl-CoA for oxidation through the citric acid cycle in the mitochondrial matrix. AceCS2 is highly expressed in the skeletal muscle and is robustly induced by fasting. Quantification of AceCS2 transcripts both in C2C12 and human myotubes indicated that fasting-induced AceCS2 gene expression appears to be independent on insulin action. Characterization of 5'-flanking region of the mouse AceCS2 gene demonstrates that Krüppel-like factor 15 (KLF15) plays a key role in the trans-activation of the AceCS2 gene. Deletion and mutation analyses of AceCS2 promoter region revealed that the most proximal KLF site is a curtail site for the trans-activation of the AceCS2 gene by KLF15. Using Sp-null Drosophila SL2 cells, we showed that the combination of KLF15 and Sp1 resulted in a synergistic activation of the AceCS2 promoter. Mutation analyses of three GC-boxes in the AceCS2 promoter indicated that the GC-box, located 8 bases downstream of the most proximal KLF15 site, is the most important GC-box in the synergistic trans-activation of the AceCS2 gene by KLF15 and Sp1. GST pull-down assays showed that KLF15 interacts with Sp1 in vitro. Quantification of various KLF transcripts revealed that 48 h fasting robustly induced the KLF15 transcripts in the skeletal muscle. Together with the trans-activation of the AceCS2 promoter, it is suggested that fasting-induced AceCS2 expression is largely contributed by KLF15. Furthermore, KLF15 overexpression induced the levels of AceCS2 transcripts both in myoblasts and in myotubes, indicating that AceCS2 gene expression in vivo is indeed induced by KLF15.

[Cortisone Acetate Reference Standard (Control 921) of National Institute of Health Sciences].

The raw material of cortisone acetate was tested for the preparation of "Cortisone Acetate Reference Standard (Control 921)". The quality of the raw material was examined and compared with the previous Cortisone Acetate Reference Standard (Control 743). Analytical data obtained were as follows: loss on drying, 0.06%; melting point, 245.9 degrees C (decomposition); optical rotation, [alpha]20D = +215.5 degrees; UV spectrum, lambda max = 239 nm and specific absorbance E 1%1 cm (239 nm) = 393; infrared spectrum, the same as that of the previous Reference Standard (Control 743); thin-layer chromatography, no impurities were detected up to 100 micrograms; high-performance liquid chromatography (HPLC), three impurities were detected; assay, 100.5% by HPLC. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Reference Standard (Control 921).

[Riboflavin Reference Standard (Control 921) of National Institute of Health Sciences].

The raw material for riboflavin was tested for preparation of the "Riboflavin Reference Standard (Control 921)". Analytical data obtained were as follows: melting point, 283.8 degrees C (decomposition); specific absorbance, E1%1 cm = 852 (267 nm), 275 (373 nm), 325 (446 nm); infrared spectrum, the same as that of JP Riboflavin Reference Standard; optical rotation [alpha]20D = 138.5 degrees; thin-layer chromatography, no impurities were detected up to 6 micrograms; high-performance liquid chromatography, a small amount of 9 impurities were detected; loss on drying, 0.16%; assay, 100.3% by spectrophotometry. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Reference Standard (Control 921).

[Ergocalciferol Reference Standard (Control 921) of National Institute of Health Sciences].

The raw material for ergocalciferol was tested for preparation of the "Ergocalciferol Reference Standard (Control 921)". Analytical data obtained were as follows: melting point, 118.1 degrees C; UV and infrared spectra, the same as those for the JP Ergocalciferol Reference Standard; specific absorbance, E1%1 cm = 457.5 (265 nm); thin-layer chromatography and high-performance liquid chromatography (HPLC), no impurities were detected; assay, 101.0% by HPLC. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Reference Standard (Control 921).

[Cholecalciferol Reference Standard (Control 921) of National Institute of Health Sciences].

The raw material for cholecalciferol was tested for preparation of the "Cholecalciferol Reference Standard (Control 921)". Analytical data obtained were as follows: melting point, 89.9 degrees C; UV and infrared spectra, the same as those for JP Cholecalciferol Reference Standard (Control 901), respectively; specific absorbance at 265 nm E1%1 cm = 472.2; optical rotation, [alpha]20D = 107.1 degrees; thin-layer chromatography and high-performance liquid chromatography (HPLC), no impurities were detected; assay, 99.97% by HPLC. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Reference Standard (Control 921).

[Pyridoxine hydrochloride reference standard (Control 911) of the National Institute of Hygienic Sciences].

The raw material of pyridoxine hydrochloride was examined for preparation of the "Pyridoxine Hydrochloride Reference Standard". Analytical data obtained were as follows: loss on drying, 0.00%; pH, 2.87; melting point, 204.6 degrees C (decomposition); infrared spectrum, same as Pyridoxine Hydrochloride Reference Standard (Control 879); thin-layer chromatography, nonimpurities were detected; assay, 100.0% by non-aqueous titration and 100.0% by UV spectrophotometry. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Standard (Control 911).

[Tocopherol reference standard (Control 911) of the National Institute of Hygienic Sciences].

The raw material for tocopherol was tested for preparation of the "Tocopherol Reference Standard (Control 911)". Analytical data obtained were as follows: infrared spectrum, same as Tocopherol Reference Standard (Control 881); absorptivity, E1%1cm (292nm) = 75.5; thin-layer chromatography, no impurities were detected up to 50.0 micrograms; high performance liquid chromatography (HPLC), four impurities were detected; assay, 100.7% by HPLC. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Standard (Control 911).

[Prednisolone reference standard (Control 911) of the National Institute of Hygienic Sciences].

Prednisolone was tested for preparation of the "Prednisolone Reference Standard (Control 911)". The quality of raw material was examined and compared with the previous Reference Standard (Control 872). Analytical data obtained were as follows: loss on drying, 0.10%; melting point, 233.2 degrees C (decomposition); optical rotation, [alpha]20D+98.77 degrees; UV spectrum, lambda max = 243nm; absorptivity, E1%1cm (243nm) = 413.5; IR spectrum, 1711, 1612, 1110, 888cm-1; one impurity was detected by thin-layer chromatography and high-performance liquid chromatography (HPLC), respectively; assay, 100.1% by HPLC. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Standard (Control 911).

[d-Camphor reference standard (Control 911) and dl-Camphor Reference Standard (Control 911) of the National Institute of Hygienic Sciences].

The raw materials of d-camphor and dl-camphor were examined for preparation of the "d-Camphor Reference Standard" and "dl-Camphor Reference Standard". Analytical data obtained were as follows: ultraviolet spectrum, lambda max = 290nm; infrared spectrum, 2958, 1742, 1045cm-1; optical rotation, [alpha]D20 = +42.7 degrees (d-camphor), [alpha]D20 = -0.3 degrees (dl-camphor); melting point, 180 degrees C (d-camphor), 179 degrees C (dl-camphor); gas-chromatography (GC), one impurity was detected in d-camphor and three impurities in dl-camphor; assay, 99.5% (d-camphor), 99.5% (dl-camphor) by GC. Based on the above results, these raw materials were authorized as the Japanese Pharmacopoeia Standard (Control 911).

[Tocopherol acetate reference standard (Control 911) of the National Institute of Hygienic Sciences].

The raw material for tocopherol acetate was tested for preparation of the "Tocopherol Acetate Reference Standard (Control 911)". Analytical data obtained were as follows: infrared spectrum, same as Tocopherol Acetate Reference Standard (Control 881); absorptivity, E1cm1% (284nm) = 43.5; thin-layer chromatography, no impurities were detected up to 50.0 micrograms; high performance liquid chromatography (HPLC), two impurities were detected; assay, 100.3% by HPLC. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Standard (Control 911).

[Diclofenamide Reference Standard (Control 891) of National Institute of Hygienic Sciences].

Quality of the raw material of diclofenamide was evaluated for preparation of "Diclofenamide Reference Standard". Analytical results for the sample were as follows: melting point 240 degrees C (decomposition); UV spectrum indicates absorption maxima at 286 and 295 nm and the absorption ratio A286/A295 1.04; IR spectrum is same as that of the USP Reference Standard; no impurity was found by TLC, but a trace of one was found by HPLC analysis; the purity is assumed to be 99.95% by HPLC analysis; loss on drying 0.1%; assay by HPLC against USP Reference Standard 100.2%. Based on the results, the present raw material was authorized to be the Reference Standard of the National Institute of Hygienic Sciences.

[Epitiostanol Reference Standard (Control 891) of National Institute of Hygienic Sciences].

The raw material of epitiostanol was examined for preparation of "Epitiostanol Reference Standard". Analytical results for the sample were as follows: melting point 130.7 degrees C (decomposition); UV spectrum indicates absorption maximum at 263 nm in ethanol; IR spectrum indicates specific absorption at 2920, 1383, 1056 and 590 cm-1; optical rotation [alpha]20D+ 24.4 degrees; no impurity was found by TLC, but a small amount of one was found by HPLC analysis and the purity is assumed to be 99.5%; water content 1.52%. Based on the results, the present raw material was authorized to be the Reference Standard of the National Institute of Hygienic Sciences.