Toshikatsu Nakabayashi

Statin-induced apoptosis linked with membrane farnesylated Ras small G protein depletion, rather than geranylated Rho protein.

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 μm), cerivastatin (3 μm), fluvastatin (3 μm), pravastatin (3 mm), or simvastatin (3 μm)) 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 μm), 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.

Evaluation of the synergistic adverse effects of concomitant therapy with statins and fibrates on rhabdomyolysis

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 ator‐vastatin‐induced myopathy, which causes little apoptosis alone. These results suggest that statins and fibrates synergistically aggravate rhabdomyolysis.

Synergistic action of statins and nitrogen-containing bisphosphonates in the development of rhabdomyolysis in L6 rat skeletal myoblasts.

Objectives Nitrogen‐containing bisphosphonates, which are widely used to treat osteoporosis, act as inhibitors of farnesyl pyrophosphate synthase, one of the key enzymes of the mevalonate pathway, and thus may have the potential to enhance the effect of statins (inhibitors of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase). In this study, we evaluated the synergistic effect of two nitrogen‐containing bisphosphonates, alendronate and risedronate, in statin‐induced apoptosis in rat skeletal L6 myoblasts.

Enzymatic and structural characterization of an archaeal thiamin phosphate synthase.

Studies on thiamin biosynthesis have so far been achieved in eubacteria, yeast and plants, in which the thiamin structure is formed as thiamin phosphate from a thiazole and a pyrimidine moiety. This condensation reaction is catalyzed by thiamin phosphate synthase, which is encoded by the thiE gene or its orthologs. On the other hand, most archaea do not seem to have the thiE gene, but instead their thiD gene, coding for a 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) kinase/HMP phosphate kinase, possesses an additional C-terminal domain designated thiN. These two proteins, ThiE and ThiN, do not share sequence similarity. In this study, using recombinant protein from the hyperthermophile archaea Pyrobaculum calidifontis, we demonstrated that the ThiN protein is an analog of the ThiE protein, catalyzing the formation of thiamin phosphate with the release of inorganic pyrophosphate from HMP pyrophosphate and 4-methyl-5-β-hydroxyethylthiazole phosphate (HET-P). In addition, we found that the ThiN protein can liberate an inorganic pyrophosphate from HMP pyrophosphate in the absence of HET-P. A structure model of the enzyme-product complex of P. calidifontis ThiN domain was proposed on the basis of the known three-dimensional structure of the ortholog of Pyrococcus furiosus. The significance of Arg320 and His341 residues for thiN-coded thiamin phosphate synthase activity was confirmed by site-directed mutagenesis. This is the first report of the experimental analysis of an archaeal thiamin synthesis enzyme.

Phosphodiesterase I in cultured cells of Mentha arvensis

Abstract Alkaline phosphodiesterase I (5′-nucleotide phosphodiesterase, EC 3.1.4.1) in cultured cells of Mentha arvensis , was purified about 75-fold by ammonium sulphate fractionation and three chromatographic steps. The optimum pH of the enzyme was 9.5. Its M r was estimated by gel filtration to be ca 105 000. The enzyme was strongly inhibited by SH reagents and HgCl 2 . It did not require divalent cations such as Mg 2+ or Ca 2+ . It hydrolysed thymidine 5′- p -nitrophenylphosphate but did not act on DNA or RNA. These properties, such as divalent cation requirement and substrate specificity, were different from those of phosphodiesterase I obtained from carrot.

Bergamottin Promotes Adipocyte Differentiation and Inhibits Tumor Necrosis Factor-α-induced Inflammatory Cytokines Induction in 3T3-L1 Cells

Nowadays, a lot of food ingredients are marketed as dietary supplements for health. Because the effectiveness and mechanisms of these compounds have not been fully characterized, they might have unknown functions. Therefore, we investigated the effect of several food ingredients (Bergamottin, Chrysin, L-Citrulline and β-Carotene) known as health foods on adipocyte differentiation by using 3T3-L1 preadipocytes. In this study, we found that Bergamottin, a furanocoumarin isolated from grapefruit juice, promotes adipocyte differentiation. In addition, Bergamottin increases the expression of adiponectin, an anti-inflammatory adipokine, and peroxisome proliferator activated receptor γ (PPARγ), a nuclear receptor regulating adipocyte differentiation. Furthermore, the anti-inflammatory activity of Bergamottin was demonstrated by its inhibition of the activation of nuclear factor-κB (NF-κB), an inflammatory transcription factor. Stimulation of mature 3T3-L1 adipocytes by tumor necrosis factor-α (TNF-α) decreased the expression of the endogeneous NF-κB inhibitor, IκBα. Treatment with Bergamottin further decreased the TNF-α-induced change in IκBα expression, suggesting that Bergamottin mediated the inhibition of NF-κB activation. In addition, Bergamottin decreased the TNF-α-induced increase in the mRNA levels of pro-inflammatory adipokines, monocyte chemoattractant protein-1 and interleukin-6. Taken together, our results show that Bergamottin treatment could inhibit inflammatory activity through promoting adipocyte differentiation, which in turn suggests that Bergamottin has the potential to minimize the risk factors of metabolic syndrome.

Inhibitory Effect of Fruit Juices on the Doxorubicin Metabolizing Activity of Carbonyl Reductase 1

BACKGROUND AND OBJECTIVE Doxorubicin, an anthracycline anti-cancer drug, is effective for breast cancer and childhood lymphoma. Chronic cardiotoxicity has been known as a critical adverse effect of doxorubicin and is attributed to its metabolite doxorubicinol produced by carbonyl reductase 1 (CBR1, SDR21C1). Some flavonoids have been reported to act as inhibitors for CBR1, therefore, commercially available juices containing flavonoids are likely to be applicable as a prophylactic treatment against doxorubicin-induced cardiotoxicity by suppression of doxorubicinol production. In the study, fruit juices containing flavonoids were investigated for inhibitory effects on CBR1. METHOD Recombinant CBR1 protein was subjected to in vitro enzymatic assays with/without juices. An apple juice and a grapefruit juice were selected in the present study as candidates capable of inhib-iting CBR1. RESULTS The enzymatic assays revealed that both juices potently inhibit the CBR1-mediated metabolic conversion of doxorubicin to doxorubicinol in concertation-dependent manner. The concentrations required for obtaining 50% inhibition (IC50) were 0.0012% (v/v) and 0.0014% (v/v) for apple and grapefruit juices, respectively. Additionally, it is worth noting that these juices showed inhibitory effects on doxorubicin metabolism by CBR1 even at very low concentrations (0.0001% (v/v)). CONCLUSION An apple juice and a grape fruit juice showed strong inhibitory effects on doxorubicin metabolism by CBR1 in vitro. These results suggest that the intake of flavonoid-containing juices can be a promising measure for protection against doxorubicin-induced cardiac toxicity, enabling patients to keep higher adherence with routine use in light of safety, economic performance and stable supply to market.

Identification of a functional antioxidant responsive element in the promoter of the Chinese hamster carbonyl reductase 3 (Chcr3) gene

CHCR3, a member of the short‐chain dehydrogenase/reductase superfamily, is a carbonyl reductase 3 enzyme in Chinese hamsters. Carbonyl reductase 3 in humans has been believed to involve the metabolism and/or pharmacokinetics of anthracycline drugs, and the mechanism underlying the gene regulation has been investigated. In this study, the nucleotide sequence of the Chcr3 promoter was originally determined, and its promoter activity was characterised. The proximal promoter region is TATA‐less and GC‐rich, similar to the promoter region of human carbonyl reductase 3. Cobalt stimulated the transcriptional activity of the Chcr3 gene. The results of a luciferase gene reporter assay demonstrated that cobalt‐induced stimulation required an antioxidant responsive element. Forced expression of Nrf2, the transcription factor that binds to antioxidant responsive elements, enhanced the transcriptional activity of the Chcr3 gene. These results suggest that cobalt induces the expression of the Chcr3 gene via the Nrf2‐antioxidant responsive element pathway.

MECHANICAL BONE PROPERTIES OF OBESE MODEL SHR/NDmcr‐cp RATS

1 High‐blood pressure or diabetes may be related to the loss of bone mass or the development of osteoporosis. We examined the mechanical bone properties of the SHR/NDmcr‐cp (SHR‐cp) rat, an obese strain that develops hypertension, hyperlipidaemia and insulin‐independent diabetes. 2 The mechanical properties of the femur of 22‐week‐old Wistar‐Kyoto (WKY) and SHR‐cp rats were measured by Pengs three‐point bending procedure modified by Shintani. Femurs were then defatted and dried. After weighing, the dried bones were ashed and the ash was weighed. The values of the dry weight, ash weight and ash weight/dry weight (%) were used as a description of the physical parameters of the bone. 3 All values of stiffness, strength, toughness and ductility in SHR‐cp were significantly lower than those of WKY rats (P < 0.05). The value of ash weight/dry weight (%) was lower in SHR‐cp rats (P < 0.01). These results showed that bone fragility was greater in SHR‐cp rats, indicative of osteopenia.

Amiodarone interaction time differences with warfarin and digoxin

Background: Amiodarone has pharmacokinetic and pharmacodynamic interactions with various therapeutic agents. The mechanism of interaction between warfarin and amiodarone is the inhibition of warfarin metabolism by amiodarone, and that between digoxin and amiodarone is the inhibition of digoxin transport by amiodarone. Objective: To investigate the pharmacokinetic magnitude of the time differences between amiodarone–warfarin and amiodarone–digoxin interactions. Methods: Amiodarone was administered concomitantly to 79 inpatients who had been receiving fixed-maintenance doses of warfarin or digoxin. Seventy-seven inpatients were prescribed warfarin therapy, and 54 inpatients were prescribed digoxin therapy. To determine serum concentrations of the warfarin enantiomers digoxin, amiodarone, and desethylamiodarone blood samples were obtained with coadministration of amiodarone. Serum S- and R-warfarin, amiodarone, and desethylamiodarone concentrations were measured by HPLC methods, and serum digoxin concentrations were measured by a fluorescence polarization immunoassay. Results: A remarkable decrease of S-warfarin clearance was observed within approximately the first 2 weeks after coadministration of amiodarone. Only a small decrease in R-warfarin clearance was observed. Digoxin clearance was gradually decreased with time, and a good reverse correlation was obtained between amiodarone or desethylamiodarone concentrations and digoxin clearance. Conclusions: Relatively short-term monitoring of warfarin clearance is required when amiodarone is coadministered. Long-term monitoring of digoxin serum amiodarone and desethylamiodarone concentrations is necessary to detect the amiodarone–digoxin interaction.